CN109609696B - Nucleic acid reagent, kit, system and method for detecting human papilloma virus - Google Patents
Nucleic acid reagent, kit, system and method for detecting human papilloma virus Download PDFInfo
- Publication number
- CN109609696B CN109609696B CN201811641953.0A CN201811641953A CN109609696B CN 109609696 B CN109609696 B CN 109609696B CN 201811641953 A CN201811641953 A CN 201811641953A CN 109609696 B CN109609696 B CN 109609696B
- Authority
- CN
- China
- Prior art keywords
- tube
- channel
- type
- fluorescent
- positive
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000701806 Human papillomavirus Species 0.000 title claims abstract description 76
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 49
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 49
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 49
- 239000012445 acidic reagent Substances 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title abstract description 35
- 239000000523 sample Substances 0.000 claims abstract description 90
- 238000001514 detection method Methods 0.000 claims abstract description 50
- 238000002844 melting Methods 0.000 claims description 177
- 230000008018 melting Effects 0.000 claims description 177
- 238000006243 chemical reaction Methods 0.000 claims description 68
- 239000007850 fluorescent dye Substances 0.000 claims description 47
- 238000003908 quality control method Methods 0.000 claims description 30
- 241000341655 Human papillomavirus type 16 Species 0.000 claims description 29
- 239000003153 chemical reaction reagent Substances 0.000 claims description 22
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 21
- 230000000694 effects Effects 0.000 claims description 7
- 239000013641 positive control Substances 0.000 claims description 7
- 238000004590 computer program Methods 0.000 claims description 6
- 239000013642 negative control Substances 0.000 claims description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 4
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 description 163
- 206010008342 Cervix carcinoma Diseases 0.000 description 14
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 14
- 201000010881 cervical cancer Diseases 0.000 description 14
- 238000012360 testing method Methods 0.000 description 14
- 238000003752 polymerase chain reaction Methods 0.000 description 12
- 238000012795 verification Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 238000012408 PCR amplification Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 208000009608 Papillomavirus Infections Diseases 0.000 description 6
- 238000003205 genotyping method Methods 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 102100030500 Heparin cofactor 2 Human genes 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- COCMHKNAGZHBDZ-UHFFFAOYSA-N 4-carboxy-3-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]benzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(C([O-])=O)=CC=C1C(O)=O COCMHKNAGZHBDZ-UHFFFAOYSA-N 0.000 description 3
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical group C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 3
- 101710153650 Heparin cofactor 2 Proteins 0.000 description 3
- 208000022361 Human papillomavirus infectious disease Diseases 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 210000005000 reproductive tract Anatomy 0.000 description 3
- 241000588626 Acinetobacter baumannii Species 0.000 description 2
- 241000726103 Atta Species 0.000 description 2
- 241000222122 Candida albicans Species 0.000 description 2
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- 102100023321 Ceruloplasmin Human genes 0.000 description 2
- 241000606153 Chlamydia trachomatis Species 0.000 description 2
- 241000194032 Enterococcus faecalis Species 0.000 description 2
- 241000701074 Human alphaherpesvirus 2 Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241000204048 Mycoplasma hominis Species 0.000 description 2
- 241000588652 Neisseria gonorrhoeae Species 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 241000589884 Treponema pallidum Species 0.000 description 2
- 241000224527 Trichomonas vaginalis Species 0.000 description 2
- 241000202921 Ureaplasma urealyticum Species 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 229940095731 candida albicans Drugs 0.000 description 2
- 229940038705 chlamydia trachomatis Drugs 0.000 description 2
- 238000013211 curve analysis Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 229940032049 enterococcus faecalis Drugs 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 210000004392 genitalia Anatomy 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000009826 neoplastic cell growth Effects 0.000 description 2
- 229960002566 papillomavirus vaccine Drugs 0.000 description 2
- 230000002085 persistent effect Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 229940124957 Cervarix Drugs 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 101710121996 Hexon protein p72 Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 101710157639 Minor capsid protein Proteins 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 101710136297 Protein VP2 Proteins 0.000 description 1
- RXUWDKBZZLIASQ-UHFFFAOYSA-N Puerarin Natural products OCC1OC(Oc2c(O)cc(O)c3C(=O)C(=COc23)c4ccc(O)cc4)C(O)C(O)C1O RXUWDKBZZLIASQ-UHFFFAOYSA-N 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 208000032124 Squamous Intraepithelial Lesions Diseases 0.000 description 1
- CUJRVFIICFDLGR-UHFFFAOYSA-N acetylacetonate Chemical compound CC(=O)[CH-]C(C)=O CUJRVFIICFDLGR-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940031416 bivalent vaccine Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 229940032077 cervical cancer vaccine Drugs 0.000 description 1
- 238000002573 colposcopy Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 208000003154 papilloma Diseases 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- HKEAFJYKMMKDOR-VPRICQMDSA-N puerarin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=CC(C2=O)=C1OC=C2C1=CC=C(O)C=C1 HKEAFJYKMMKDOR-VPRICQMDSA-N 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000005582 sexual transmission Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 229940031351 tetravalent vaccine Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/708—Specific hybridization probes for papilloma
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present disclosure relates to a nucleic acid reagent, a kit, a system and a method for detecting human papilloma virus, wherein the nucleic acid reagent comprises primers shown in SEQ ID No.1-50 and probes shown in SEQ ID No.53-77, which are respectively stored independently or randomly mixed with each other. According to the nucleic acid reagent, the kit, the system and the method for detecting the human papilloma virus, which are established by the primers and the probes, the rapid, comprehensive, sensitive, specific and automatic detection result judgment can be realized.
Description
Technical Field
The present disclosure relates to the field of biotechnology, and in particular, to a nucleic acid reagent, a kit, a system and a method for detecting human papilloma virus.
Background
With the popularity of screening, the incidence and mortality of cervical cancer has decreased significantly in developed countries or regions, with a 33% reduction in cervical cancer incidence from 1991 to 2000 in some developed countries such as the united kingdom and australia. Cervical cancer is the second most common malignancy in women, and mortality remains high, due to underdeveloped area screening that is not performed or regulated worldwide. The etiology of cervical cancer is clear, and a large number of researches indicate that persistent infection of high-risk Human Papilloma Virus (HPV) is a main pathogenic factor of cervical cancer and precancerous lesion.
HPV belongs to the genus Papovavirdae (Papovavirdae) papilloma virus, and primarily infects humans by direct or indirect contact with contaminating materials or by sexual transmission. HPV not only has host specificity, but also has tissue specificity, and can only infect skin and mucosal epithelial cells of human, causing various papillomas or warts of human skin and epithelial proliferative injuries of mucosal genital tract. Persistent HPV infection is a major cause of cervical cancer and some genital neoplasias.
HPV is a spherical, non-enveloped, double-stranded DNA virus. The nucleocapsid is a symmetrical 20-hedron and consists of 72 capsomeres, and the capsomeres consist of two capsid proteins of L1 and L2. The HPV genome is 8000bp Long, encodes 8 main Open Reading Frames (ORF), and is divided into 3 functional regions, namely an early transcription Region (E Region), a late transcription Region (L Region) and a Long Control Region (LCR). The E region codes early proteins such as E1, E2, E4, E5, E6, E7 and the like, and is involved in the functions of replication, transcription, translation regulation, transformation and the like of the virus; region L encodes a major capsid protein and a minor capsid protein. L1 accounts for about 80% of capsid protein, and is highly conserved, with less L2 content and more variation. LCR contains the replication origin of HPV genomic DNA and the regulatory elements required for HPV expression, regulating the transcription and replication of the virus.
At present, more than 100 HPV genotypes are available, and nearly 33% of the genotypes are related to genital tract injury. The infection of the reproductive tract can be divided into two categories: low Risk (LR) and High Risk (HR). Wherein the HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56,58, 59 and 68 are common HPV high-risk type infections and are closely related to cervical cancer; and HPV6 and 11 are low-risk types of HPV. HPV16 and 18 are most common among the high risk types of HPV, and investigation make internal disorder or usurp indicated that 70% of cervical cancers worldwide were associated with HPV16 and 18 infections. Whereas HPV6 and 11 are associated with most genital neoplasias. HPV genotyping is clinically significant. Persistent HPV high-risk infection can progress to cervical cancer, and several studies have shown that different high-risk HPV genotypes have different carcinogenic risks. According to the annual American Society for vaginoscope and Cervical Pathology (ASCCP) guidelines for Cervical cancer screening, the strategy for screening women infected with HPV16 and 18 is different from that for other high-risk types of HPV infection, and women aged 30 years old or older who are infected with HPV16 and 18 should be directly subjected to colposcopic examination, while other high-risk types are positive, cytology and HPV detection can be repeated after 12 months, and the Colposcopy examination is performed only if the detection is positive again. In addition, HPV vaccines are used for preventing cervical cancer, and tetravalent vaccines produced by Merk corporation and bivalent vaccines Cervarix produced by puerarin smith corporation are currently on the market. HPV genotyping assays are performed both before and after vaccination. HPV genotyping is also of great value for monitoring HPV vaccine use.
Since HPV cannot be cultured in traditional cell culture media, other classical direct virological diagnostic techniques, such as electron microscopy and immunohistochemistry, are poorly sensitive and specific for routine HPV detection. Therefore, all current HPV detection reagents (including self-made or commercial reagents) for diagnosis rely mainly on viral nucleic acid detection. HPV nucleic acid detection reagents can be roughly classified into three types: (1) a universal detection reagent for HR-HPV nucleic acid genotypes, which only reports whether HR-HPV infection exists, but does not report specific HPV genotypes, and HC2(Hybrid Capture 2) is the most typical representative; (2) and (3) while carrying out universal detection on the HR-HPV nucleic acid genotype, carrying out retesting on the HPV 16/18 genotype: the detection reagent reports the presence of HR-HPV infection and simultaneously reports the presence of HPV 16/18 infection. Some reagents detect infection with HPV 16/18 simultaneously with HR-HPV, others first detect the presence of HR-HPV infection and if positive, the sample is retested for the presence of infection, e.g., Cervista HPV 16/18(Hologic, Danbury, Ct, USA). (3) HPV genotyping reagent, the detection reagent can distinguish HPV specific genotypes, and directly report the result of infection of each genotype. Most of HPV genotyping reagents developed by reagent manufacturers at present belong to the same type, and many HPV nucleic acid detections developed in clinical laboratories are also adopted.
The HPV infection rate is improved along with the beginning of sexual life, and is mostly mixed infection, and various types of HPV can be simultaneously detected in more than 12-28.9%, even 85% of cervical cancer cases. At present, the main methods for clinically detecting HPV DNA in China comprise a real-time fluorescence Polymerase Chain Reaction (PCR) method, a gene chip method and a Hybrid Capture method (Hybrid Capture II, HC-II). These 3 methods are superior and inferior as a means of clinical cervical cancer screening.
At present, the domestic gene chip HPV detection reagent adopts a DNA chip technology combining PCR in-vitro amplification and DNA reverse dot hybridization. The detection result of the gene chip method can only carry out qualitative analysis on the sample to judge whether the detection site has a signal or not, and the strength of the signal point can not provide a reference in the quantitative aspect. In addition, the gene chip method has one more step of hybridization and color development than real-time PCR, so the operation steps are still relatively more complicated than real-time fluorescence PCR. After PCR amplification, a cover needs to be opened for hybridization experiments, so that the risk of PCR product pollution in a laboratory is increased, and false positive of experimental results is possibly caused.
The HC-II technology is actually a comprehensive application of nucleic acid hybridization, immunoreaction and chemiluminescence technology, has the HPV grouping (high-risk group and low-risk group) function but does not have the accurate typing function, and the method is widely used for screening and follow-up of cervical cancer. HC-II found that the sensitivity of High Squamous Intraepithelial Lesions (HSIL) was 95% and was significantly better than that of liquid-based cytology, but the specificity was 85% and slightly lower than that of liquid-based cytology. Since this method was developed abroad and brought into the market early, other methodologies developed later in China basically compare and verify with this methodology clinically for HPV DNA detection reagents. Although the standard of HPV diagnosis gold cannot be formed, a valuable reference system is provided for the development of HPV diagnostic reagents in China later. However, with the wide application at home and abroad in recent years, some clinical defects and problems are gradually exposed, such as easy generation of cross contamination, high cost, certain cross reaction between high-risk types and low-risk types, and individual high-risk type omission factor reported in foreign documents.
Disclosure of Invention
The purpose of the present disclosure is to provide a nucleic acid reagent, a kit, a system and a method for rapid, accurate and integrated typing detection of human papillomavirus.
To achieve the above object, a first aspect of the present disclosure: provided is a nucleic acid reagent for detecting human papillomavirus, wherein the nucleic acid reagent comprises primers shown in SEQ ID NO.1-50 and probes shown in SEQ ID NO.53-77, which are respectively stored independently of each other or randomly mixed with each other.
Optionally, the amount of the primer represented by SEQ ID NO.2-50 is 0.1-0.5. mu.M, 0.5-1.0. mu.M, 0.1-0.5. mu.M, 0.5-1.0.5. mu.M, 0.5-0.5. mu.0.5. mu.M, 0.5-0.0.5. mu.0.5. mu.0, 0.5-0.5. mu.M, 0.5-0.5. mu.0.5. mu.0.M, 0.5-0.5. mu.0.5. mu.0, 0.5-0.5. mu.0, 0.5. mu.0.M, 0.5-0.5. mu.0.5. mu.0, 0.5 to 0.0.5. mu.M, 0.0.0 to 0.5. mu.5. mu.0.5 to 0.0.5. mu.M, 0.0.0.0 to 0 to 0.M, 0.5 to 0.0.0.0.0.0.5 to 0.5 to 0.0.0.5 to 0.0.5. mu.0.5 to 0.0.0.5. mu.0.0.M, 0.5 to 0 to 0.5 to 0 to 0.0.0.M, 0.0 to 0.0.0 to 0 to 1 to 0.0.0.0.5 to 0 to 0.0.0.5 to 0.0.0.0.M, 0.5 to 0.0 to 0.0.0.0.0 to 0 to 0.5 μ M, 0 to 0.5 μ M, 0.0.0.0.5 μ M, 0.0.5 μ M, 0 to 0.0.5 μ M, 0 to 0.5 μ M, 0 to 0.0.0 to 1 to 0.0.0.0.0 to 0 to 0.5 μ M, 0 to 0.0.0 to 0.0.0.0 to 0.0.0 to 0 to 0.5 μ M, 0.0.5 μ M, 0.0.0.5 to 1 to 0.0.0 to 0 to 0.5 μ M, 0 to 0.0 to 0 to 0.5 μ M, 0 to 0.0 to 0 to 0.5 μ M, 0 to 0.0 to 0 to 0.5 to 0 to 0.0.0 to 1 to 0.5 μ M, 0 to 0.0 to 0 to 0.5 μ M, 0, 0.1 to 0.5. mu.M, 0.5 to 1.0. mu.M, 0.1 to 0.5. mu.M, 0.5 to 1.0. mu.M and 0.1 to 0.5. mu.M, and the content of each of the probes represented by SEQ ID nos. 53 to 77 is 0.1 to 0.3. mu.M.
Optionally, the nucleic acid reagent further comprises a positive internal quality control;
the positive endoplasmic control contains a primer shown by SEQ ID NO.51-52 and a probe shown by SEQ ID NO. 78.
Optionally, the nucleic acid reagent comprises tube a, tube B, tube C, and tube D; the tube A contains primers shown as SEQ ID NO.1-2,5-6,9-12,19-20,27-28,43-44,51-52 and probes shown as SEQ ID NO.53,55,57-58,62,66,74, 78; the tube B comprises primers shown as SEQ ID NO.3-4,7-8,13-14,21-22,29-32,45-46 and 51-52 and probes shown as SEQ ID NO.54,56,59,63,67,68,75 and 78; the C tube comprises primers shown in SEQ ID NO.7-8,15-16,23-24,29-30,33-34,39-40,47-48 and 51-52 and probes shown in SEQ ID NO.56,60,64,67,69,72,76 and 78; the D tube contains primers shown in SEQ ID NO.5-6,17-18,25-26,35-38,41-42,49-50,51-52 and probes shown in SEQ ID NO.55,61,65,70,71,73,77 and 78.
Alternatively, the probes shown in SEQ ID NO.55-56,58-61 have a first fluorescent label; the probes shown in SEQ ID NO.57,62-65,67,70 have a second fluorescent label; the probes shown in SEQ ID NO.53,54,66,68-69,71-73 have a third fluorescent label; the probes shown in SEQ ID NO.74-78 have a fourth fluorescent label; the first fluorescent label, the second fluorescent label, the third fluorescent label and the fourth fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a JOE fluorescent label, a TAMRA fluorescent label, a CY5 fluorescent label, a ROX fluorescent label and a Quasar670 fluorescent label.
Optionally, the human papillomavirus comprises at least one of HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type, and HPV83 type.
In a second aspect of the present disclosure: there is provided a kit for detecting human papillomavirus, the kit containing the nucleic acid reagents according to the first aspect of the present disclosure, and optionally, the kit further contains at least one of a reaction system buffer, a DNA polymerase, magnesium ions, dntps, and water.
A third aspect of the disclosure: there is provided use of the nucleic acid reagent according to the first aspect of the present disclosure in the preparation of a kit for detecting human papillomavirus.
A fourth aspect of the present disclosure: providing a system for detecting human papillomavirus, the system comprising a PCR instrument having an a-tube detector, a B-tube detector, a C-tube detector and a D-tube detector, the a-tube detector, the B-tube detector, the C-tube detector and the D-tube detector being nucleic acid reagent storage containers loaded with nucleic acid reagents as described above, respectively, the PCR instrument comprising a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel, the first fluorescence channel, the second fluorescence channel, the third fluorescence channel and the fourth fluorescence channel being different and each independently being a FAM fluorescence channel, a JOE fluorescence channel, a TAMRA fluorescence channel, a CY5 fluorescence channel, a ROX fluorescence channel or a Quasar670 fluorescence channel; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
if the positive control and the negative control are established, the detection result is valid;
judging the first fluorescent channel of the tube A to be positive by HPV16 if the first fluorescent channel of the tube A has a melting peak curve with a Tm value of 66 ℃, judging the first fluorescent channel of the tube A to be positive by HPV31 if the first fluorescent channel of the tube A has a melting peak curve with a Tm value of 58 ℃, judging the second fluorescent channel of the tube A to be positive by HPV26 if the second fluorescent channel of the tube A has a melting peak curve with a Tm value of 65 ℃, judging the second fluorescent channel of the tube A to be positive by HPV42 if the second fluorescent channel of the tube A has a melting peak curve with a Tm value of 55 ℃, judging the third fluorescent channel of the tube A to be positive by HPV51 if the third fluorescent channel of the tube A has a melting peak curve with a Tm value of 56 ℃, judging the third fluorescent channel of the tube A to be positive by HPV6 if the fourth fluorescent channel of the tube A has a melting peak curve with a Tm value of 63 ℃, and judging the fourth fluorescent channel of the tube A to be qualified for internal quality control if the fourth fluorescent channel of the tube A has a melting peak curve with a Tm value of 52 ℃;
judging the first fluorescent channel of the B tube to be positive by HPV18 if the first fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 67 ℃, judging the first fluorescent channel of the B tube to be positive by HPV33 if the first fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 54 ℃, judging the second fluorescent channel of the B tube to be positive by HPV52 if the second fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 69 ℃, judging the second fluorescent channel of the B tube to be positive by HPV43 if the second fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 56 ℃, judging the third fluorescent channel of the B tube to be positive by HPV53 if the third fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 63 ℃, judging the third fluorescent channel of the B tube to be positive by HPV11 if the third fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 51 ℃, judging the fourth fluorescent channel of the B tube to be positive by HPV81, and judging the fourth fluorescent channel of the B tube to be positive by internal quality control;
judging that the HPV18 is positive if the melting peak curve corresponding to the Tm value of 67 ℃ exists in the first fluorescent channel of the C tube, judging that the HPV35 is positive if the melting peak curve corresponding to the Tm value of 56 ℃ exists in the first fluorescent channel of the C tube, judging that the HPV52 is positive if the melting peak curve corresponding to the Tm value of 69 ℃ exists in the second fluorescent channel of the C tube, judging that the HPV44 is positive if the melting peak curve corresponding to the Tm value of 54 ℃ exists in the second fluorescent channel of the C tube, judging that the HPV56 is positive if the melting peak curve corresponding to the Tm value of 62 ℃ exists in the third fluorescent channel of the C tube, judging that the HPV66 is positive if the melting peak curve corresponding to the Tm value of 53 ℃ exists in the third fluorescent channel of the C tube, judging that the sample is PV82 positive if the melting peak curve corresponding to the Tm value of 61 ℃ exists in the fourth fluorescent channel of the C tube, and judging that the internal quality control is qualified if the melting peak curve corresponding to the Tm value of 52 ℃ exists in the fourth fluorescent channel of the C tube;
if the D tube first fluorescence channel has a melting peak curve with a Tm value of 66 ℃, the D tube first fluorescence channel is judged to be HPV16 positive, if the D tube first fluorescence channel has a melting peak curve with a Tm value of 57 ℃, the D tube second fluorescence channel has a melting peak curve with a Tm value of 65 ℃, the D tube second fluorescence channel is judged to be HPV58 positive, if the D tube second fluorescence channel has a melting peak curve with a Tm value of 55 ℃, the D tube second fluorescence channel is judged to be HPV45 positive, if the D tube third fluorescence channel has a melting peak curve with a Tm value of 60 ℃, the D tube third fluorescence channel is judged to be HPV59 positive, if the D tube third fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D tube third fluorescence channel is judged to be HPV68 positive, if the D tube fourth fluorescence channel has a melting peak curve with a Tm value of 62 ℃, the D tube fourth fluorescence channel is judged to be HPV83 positive, and if the D tube fourth fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D tube fourth fluorescence channel is judged to be positive internal quality control qualified.
The fifth aspect of the present disclosure: there is provided a method for detecting human papillomavirus, wherein the method comprises: carrying out PCR amplification on DNA of a sample to be detected by adopting the nucleic acid reagent; the PCR instrument for carrying out the PCR amplification comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel; the first, second, third, and fourth fluorescent channels are different from each other and are each independently a FAM, JOE, TAMRA, CY5, ROX, or Quasar670 fluorescent channel; and the following discrimination is made:
if the positive control and the negative control are established, the detection result is valid;
judging the first fluorescent channel of the tube A to be positive by HPV16 if the first fluorescent channel of the tube A has a melting peak curve with a Tm value of 66 ℃, judging the first fluorescent channel of the tube A to be positive by HPV31 if the first fluorescent channel of the tube A has a melting peak curve with a Tm value of 58 ℃, judging the second fluorescent channel of the tube A to be positive by HPV26 if the second fluorescent channel of the tube A has a melting peak curve with a Tm value of 65 ℃, judging the second fluorescent channel of the tube A to be positive by HPV42 if the second fluorescent channel of the tube A has a melting peak curve with a Tm value of 55 ℃, judging the third fluorescent channel of the tube A to be positive by HPV51 if the third fluorescent channel of the tube A has a melting peak curve with a Tm value of 56 ℃, judging the third fluorescent channel of the tube A to be positive by HPV6 if the fourth fluorescent channel of the tube A has a melting peak curve with a Tm value of 63 ℃, and judging the fourth fluorescent channel of the tube A to be qualified for internal quality control if the fourth fluorescent channel of the tube A has a melting peak curve with a Tm value of 52 ℃;
judging the first fluorescent channel of the B tube to be positive by HPV18 if the first fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 67 ℃, judging the first fluorescent channel of the B tube to be positive by HPV33 if the first fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 54 ℃, judging the second fluorescent channel of the B tube to be positive by HPV52 if the second fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 69 ℃, judging the second fluorescent channel of the B tube to be positive by HPV43 if the second fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 56 ℃, judging the third fluorescent channel of the B tube to be positive by HPV53 if the third fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 63 ℃, judging the third fluorescent channel of the B tube to be positive by HPV11 if the third fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 51 ℃, judging the fourth fluorescent channel of the B tube to be positive by HPV81, and judging the fourth fluorescent channel of the B tube to be positive by internal quality control;
judging that the HPV18 is positive if the melting peak curve corresponding to the Tm value of 67 ℃ exists in the first fluorescent channel of the C tube, judging that the HPV35 is positive if the melting peak curve corresponding to the Tm value of 56 ℃ exists in the first fluorescent channel of the C tube, judging that the HPV52 is positive if the melting peak curve corresponding to the Tm value of 69 ℃ exists in the second fluorescent channel of the C tube, judging that the HPV44 is positive if the melting peak curve corresponding to the Tm value of 54 ℃ exists in the second fluorescent channel of the C tube, judging that the HPV56 is positive if the melting peak curve corresponding to the Tm value of 62 ℃ exists in the third fluorescent channel of the C tube, judging that the HPV66 is positive if the melting peak curve corresponding to the Tm value of 53 ℃ exists in the third fluorescent channel of the C tube, judging that the sample is PV82 positive if the melting peak curve corresponding to the Tm value of 61 ℃ exists in the fourth fluorescent channel of the C tube, and judging that the internal quality control is qualified if the melting peak curve corresponding to the Tm value of 52 ℃ exists in the fourth fluorescent channel of the C tube;
if the D tube first fluorescence channel has a melting peak curve with a Tm value of 66 ℃, the D tube first fluorescence channel is judged to be HPV16 positive, if the D tube first fluorescence channel has a melting peak curve with a Tm value of 57 ℃, the D tube second fluorescence channel has a melting peak curve with a Tm value of 65 ℃, the D tube second fluorescence channel is judged to be HPV58 positive, if the D tube second fluorescence channel has a melting peak curve with a Tm value of 55 ℃, the D tube second fluorescence channel is judged to be HPV45 positive, if the D tube third fluorescence channel has a melting peak curve with a Tm value of 60 ℃, the D tube third fluorescence channel is judged to be HPV59 positive, if the D tube third fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D tube third fluorescence channel is judged to be HPV68 positive, if the D tube fourth fluorescence channel has a melting peak curve with a Tm value of 62 ℃, the D tube fourth fluorescence channel is judged to be HPV83 positive, and if the D tube fourth fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D tube fourth fluorescence channel is judged to be positive internal quality control qualified.
The beneficial effect of this disclosure lies in:
the method disclosed by the invention is used for detecting the human papilloma virus by the ParaDNA and Hybeacon probe technology in a typing manner, avoids the long operation time and complicated operation of smear, culture, RT-PCR detection and other methods, realizes quick, simple, convenient, sensitive and specific automatic result judgment, and achieves the following detection effects:
single tube multiplex detection
The present disclosure can detect human papillomaviruses including HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type in a one-time typing way, the detection flow is simple, the result is automatically interpreted and reliable, and the time, labor and reagent costs are saved.
(II) convenient operation
The clinical sample can be directly detected by a sampler matched with a ParaDNA system, so that a reliable result can be obtained, the sample extraction step which is expensive and time-consuming is avoided, and the emergency detection except a professional laboratory is realized.
(III) integration solution
The method provides a set of comprehensive, rapid, accurate and simple and convenient integrated solution for the detection of the HPV Chinese epidemic subtypes, and comprises the steps of rapid extraction of nucleic acid, fluorescent PCR amplification and automatic result judgment.
(IV) high sensitivity
The method can realize qualitative detection of 25 HPV subtypes, and the detection sensitivity of each target gene in a reaction system can reach 103copy/mL, comparable to the sensitivity of a single real-time fluorescent PCR assay.
(V) good specificity
In the nucleic acid reagent disclosed by the invention, all primers are subjected to BLAST comparison analysis, have high conservative property and specificity, can distinguish detection targets from each other, and can also distinguish from other similar bacteria with the same species and living environment, such as human papilloma virus HPV 40 type, HPV54 type, HPV61 type, HPV67 type, HPV69 type, HPV70 type, HPV71 type and HPV72 type, herpes simplex virus II type, treponema pallidum, ureaplasma urealyticum, mycoplasma hominis, Neisseria gonorrhoeae, Candida albicans, trichomonas vaginalis, chlamydia trachomatis, Acinetobacter baumannii and enterococcus faecalis.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
The first aspect of the disclosure: provided is a nucleic acid reagent for detecting human papillomavirus, wherein the nucleic acid reagent comprises primers shown in SEQ ID NO.1-50 and probes shown in SEQ ID NO.53-77, which are respectively stored independently of each other or randomly mixed with each other.
The method detects the human papillomavirus by using the ParaDNA and Hybeacon probe technology, avoids long operation time and complicated operation of smear, culture, RT-PCR detection and other methods, and can quickly, accurately and integrally detect the human papillomaviruses including HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type.
The Hybeacon probe technology has high requirements on the probe, and the Tm value of the probe is particularly important; in addition, the effect of the combination of the probe and the primer also has an important influence on the amplification effect. In the design process of the primers and the probes, the problem of co-amplification of the primers and the probes of different target genes in a reaction system is considered, namely, the Tm value, the difference value of the Tm values of the probes corresponding to the targets, GC content and the like are evaluated, the conditions of hairpin structures, dimers and the like are avoided, the alternative primers and the probe segments can be ensured to respectively and comprehensively cover the enteroviruses, and the primers and the probes have good specificity and high coverage.
Further, the amount of the primer represented by SEQ ID NO.2-50 may be 0.1-0.5. mu.M, 0.5-1.0. mu.M, 0.1-0.5. mu.M, 0.5-1.0.0.5. mu.0, 0.5-0.5. mu.0. mu.M, 0.5-0.5. mu.0, 0.5. mu.5-0.M, 0.5-0.5. mu.0, 0.5. mu.5-0, 0.5-0.5. mu.0, 0.5. mu.5-0.M, 0.0.5-0.5. mu.0.5, 0.M, 0.5-0.5. mu.5-0.5 to 0.0.5. mu.0.5. mu.M, 0.0.0.5 to 0.M, 0.5 to 0.M, 0.0.0.0.0.5 to 0.5 to 0.0.0.0.5 to 0.0.5 to 0.0.0.5 to 0.0.0.M, 0.5 to 1 to 0.5 to 0 to 0.0.0.0.0.0 to 0.0.0.0.0 to 1 to 0 to 1 to 0.5 μ M, 0.0.5 to 0 to 0.0.0.5 to 0.0.0.0.0.5 to 0.5 μ M, 0 to 0.0.0.0 to 1 to 0.5 μ M, 0 to 0.5 μ M, 0.0.0.0.0.5 μ M, 0.0.5 μ M, 0 to 0.0 to 0 to 1 to 0.0.5 μ M, 0.5 μ M, 0.0.5 μ M, 0.0.0 to 0 to 0.5 μ M, 0.0.0 to 1 to 0.0.0.0.0.0 to 0 to 0.0 to 0 to 1 to 0.0.0 to 0 to 0.0.5 μ M, 0 to 0.0 to 1 to 0 to 1 to 0.0.0.0.5 μ M, 0 to 1 to 0.0 to 0 to 0.5 μ M, 0.0.0 to 0.0.5 μ M, 0.5 μ M, 0.0.5 μ M, 0.0 to 0.5 μ M, 0 to 0.0 to 0 to 1 to 0 to 1 to 0.5 μ M, 0.0.0 to 1 to 0.5 μ M, 0.0.0.5 to 0 to 1 to, 0.5 to 1.0. mu.M, 0.1 to 0.5. mu.M, 0.5 to 1.0. mu.M and 0.1 to 0.5. mu.M, and the content of the probes represented by SEQ ID nos. 53 to 77 may be 0.1 to 0.3. mu.M each independently.
According to the present disclosure, the nucleic acid reagent may further include a positive internal quality control for the sake of quality control. Further, the positive internal quality control can contain primers shown in SEQ ID NO.51-52 and a probe shown in SEQ ID NO. 78. In this case, the content of the primers shown by SEQ ID NO.51 to 52 may be 0.5 to 1.0. mu.M and 0.1 to 0.5. mu.M, respectively, and the content of the probe shown by SEQ ID NO.78 may be 0.1 to 0.3. mu.M, respectively, relative to 1. mu.M of the primer shown by SEQ ID NO. 1. By adding the positive internal quality control, false negative detection results caused by misoperation, PCR inhibitors and the like can be effectively prompted.
According to the present disclosure, in order to enhance the accuracy of the detection result, the nucleic acid reagent may be divided into four tubes, i.e., the nucleic acid reagent may include a tube a, a tube B, a tube C, and a tube D; the tube A contains primers shown as SEQ ID NO.1-2,5-6,9-12,19-20,27-28,43-44,51-52 and probes shown as SEQ ID NO.53,55,57-58,62,66,74, 78; the tube B comprises primers shown as SEQ ID NO.3-4,7-8,13-14,21-22,29-32,45-46 and 51-52 and probes shown as SEQ ID NO.54,56,59,63,67,68,75 and 78; the C tube comprises primers shown in SEQ ID NO.7-8,15-16,23-24,29-30,33-34,39-40,47-48 and 51-52 and probes shown in SEQ ID NO.56,60,64,67,69,72,76 and 78; the D tube contains primers shown in SEQ ID NO.5-6,17-18,25-26,35-38,41-42,49-50,51-52 and probes shown in SEQ ID NO.55,61,65,70,71,73,77 and 78.
Further, the arrangement and combination of the fluorescent labels may be performed according to the respective Tm values of the probes, so that the amplification of different probes in the same system can be recognized separately. For example, as one embodiment, the probes shown in SEQ ID Nos. 55-56,58-61 have a first fluorescent label; the probes shown in SEQ ID NO.57,62-65,67,70 have a second fluorescent label; the probes shown in SEQ ID NO.53,54,66,68-69,71-73 have a third fluorescent label; the probes shown in SEQ ID NO.74-78 have a fourth fluorescent label; the first fluorescent label, the second fluorescent label, the third fluorescent label and the fourth fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a JOE fluorescent label, a TAMRA fluorescent label, a CY5 fluorescent label, a ROX fluorescent label and a Quasar670 fluorescent label. As a particularly preferred embodiment, the probes shown in SEQ ID Nos. 55 to 56,58 to 61 have FAM fluorescent labels; the probes shown as SEQ ID NO.57,62-65,67 and 70 have JOE fluorescent labels; the probes shown in SEQ ID NO.53,54,66,68-69,71-73 have TAMRA fluorescent labels; the probes shown in SEQ ID NO.74-78 have the CY5 fluorescent label. In order to enhance the peak effect, the target probe may be a double-labeled probe. In the probe, FAM is 6-carboxyfluorescein, JOE is 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein, TAMRA is 6-carboxytetramethylrhodamine, and CY5 is 5H-indocyanine.
According to the present disclosure, the human papillomavirus may include at least one of HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type, and HPV83 type.
In a second aspect of the present disclosure: there is provided a kit for detecting human papillomavirus, the kit containing the nucleic acid reagents according to the first aspect of the present disclosure, and optionally, the kit further contains at least one of a reaction system buffer, a DNA polymerase, magnesium ions, dntps, and water.
The kit disclosed by the invention can realize rapid, accurate, sensitive, specific and automatic detection result judgment, and remarkably improves the sensitivity, specificity and simplicity of simultaneously detecting human papillomaviruses including HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type.
A third aspect of the disclosure: there is provided use of the nucleic acid reagent according to the first aspect of the present disclosure in the preparation of a kit for detecting human papillomavirus.
A fourth aspect of the present disclosure: providing a system for detecting human papillomavirus, the system comprising a PCR instrument having an A-tube detector, a B-tube detector, a C-tube detector and a D-tube detector, a computing device and an output device, the A-tube detector, the B-tube detector, the C-tube detector and the D-tube detector being respectively nucleic acid reagent storage containers loaded with the above nucleic acid reagents including the A-tube, the B-tube, the C-tube and the D-tube, the PCR instrument comprising a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel, the first fluorescence channel, the second fluorescence channel, the third fluorescence channel and the fourth fluorescence channel being different and each independently being a FAM fluorescence channel, a JOE fluorescence channel, a TAA fluorescence channel, a CY5 fluorescence channel, a ROX fluorescence channel or a Quasar670 fluorescence channel; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
if the positive control and the negative control are established, the detection result is valid;
judging the first fluorescent channel of the tube A to be positive by HPV16 if the first fluorescent channel of the tube A has a melting peak curve with a Tm value of 66 ℃, judging the first fluorescent channel of the tube A to be positive by HPV31 if the first fluorescent channel of the tube A has a melting peak curve with a Tm value of 58 ℃, judging the second fluorescent channel of the tube A to be positive by HPV26 if the second fluorescent channel of the tube A has a melting peak curve with a Tm value of 65 ℃, judging the second fluorescent channel of the tube A to be positive by HPV42 if the second fluorescent channel of the tube A has a melting peak curve with a Tm value of 55 ℃, judging the third fluorescent channel of the tube A to be positive by HPV51 if the third fluorescent channel of the tube A has a melting peak curve with a Tm value of 56 ℃, judging the third fluorescent channel of the tube A to be positive by HPV6 if the fourth fluorescent channel of the tube A has a melting peak curve with a Tm value of 63 ℃, and judging the fourth fluorescent channel of the tube A to be qualified for internal quality control if the fourth fluorescent channel of the tube A has a melting peak curve with a Tm value of 52 ℃;
judging the first fluorescent channel of the B tube to be positive by HPV18 if the first fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 67 ℃, judging the first fluorescent channel of the B tube to be positive by HPV33 if the first fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 54 ℃, judging the second fluorescent channel of the B tube to be positive by HPV52 if the second fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 69 ℃, judging the second fluorescent channel of the B tube to be positive by HPV43 if the second fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 56 ℃, judging the third fluorescent channel of the B tube to be positive by HPV53 if the third fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 63 ℃, judging the third fluorescent channel of the B tube to be positive by HPV11 if the third fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 51 ℃, judging the fourth fluorescent channel of the B tube to be positive by HPV81, and judging the fourth fluorescent channel of the B tube to be positive by internal quality control;
judging that the HPV18 is positive if the melting peak curve corresponding to the Tm value of 67 ℃ exists in the first fluorescent channel of the C tube, judging that the HPV35 is positive if the melting peak curve corresponding to the Tm value of 56 ℃ exists in the first fluorescent channel of the C tube, judging that the HPV52 is positive if the melting peak curve corresponding to the Tm value of 69 ℃ exists in the second fluorescent channel of the C tube, judging that the HPV44 is positive if the melting peak curve corresponding to the Tm value of 54 ℃ exists in the second fluorescent channel of the C tube, judging that the HPV56 is positive if the melting peak curve corresponding to the Tm value of 62 ℃ exists in the third fluorescent channel of the C tube, judging that the HPV66 is positive if the melting peak curve corresponding to the Tm value of 53 ℃ exists in the third fluorescent channel of the C tube, judging that the sample is PV82 positive if the melting peak curve corresponding to the Tm value of 61 ℃ exists in the fourth fluorescent channel of the C tube, and judging that the internal quality control is qualified if the melting peak curve corresponding to the Tm value of 52 ℃ exists in the fourth fluorescent channel of the C tube;
if the D tube first fluorescence channel has a melting peak curve with a Tm value of 66 ℃, the D tube first fluorescence channel is judged to be HPV16 positive, if the D tube first fluorescence channel has a melting peak curve with a Tm value of 57 ℃, the D tube second fluorescence channel has a melting peak curve with a Tm value of 65 ℃, the D tube second fluorescence channel is judged to be HPV58 positive, if the D tube second fluorescence channel has a melting peak curve with a Tm value of 55 ℃, the D tube second fluorescence channel is judged to be HPV45 positive, if the D tube third fluorescence channel has a melting peak curve with a Tm value of 60 ℃, the D tube third fluorescence channel is judged to be HPV59 positive, if the D tube third fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D tube third fluorescence channel is judged to be HPV68 positive, if the D tube fourth fluorescence channel has a melting peak curve with a Tm value of 62 ℃, the D tube fourth fluorescence channel is judged to be HPV83 positive, and if the D tube fourth fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D tube fourth fluorescence channel is judged to be positive internal quality control qualified.
The fifth aspect of the present disclosure: there is provided a method for detecting human papillomavirus, wherein the method comprises: performing PCR amplification on DNA of a sample to be detected by adopting the nucleic acid reagent comprising the tube A, the tube B, the tube C and the tube D; the PCR instrument for carrying out the PCR amplification comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel; the first, second, third, and fourth fluorescent channels are different from each other and are each independently a FAM, JOE, TAMRA, CY5, ROX, or Quasar670 fluorescent channel; and the following discrimination is made:
if the positive control and the negative control are established, the detection result is valid;
judging the first fluorescent channel of the tube A to be positive by HPV16 if the first fluorescent channel of the tube A has a melting peak curve with a Tm value of 66 ℃, judging the first fluorescent channel of the tube A to be positive by HPV31 if the first fluorescent channel of the tube A has a melting peak curve with a Tm value of 58 ℃, judging the second fluorescent channel of the tube A to be positive by HPV26 if the second fluorescent channel of the tube A has a melting peak curve with a Tm value of 65 ℃, judging the second fluorescent channel of the tube A to be positive by HPV42 if the second fluorescent channel of the tube A has a melting peak curve with a Tm value of 55 ℃, judging the third fluorescent channel of the tube A to be positive by HPV51 if the third fluorescent channel of the tube A has a melting peak curve with a Tm value of 56 ℃, judging the third fluorescent channel of the tube A to be positive by HPV6 if the fourth fluorescent channel of the tube A has a melting peak curve with a Tm value of 63 ℃, and judging the fourth fluorescent channel of the tube A to be qualified for internal quality control if the fourth fluorescent channel of the tube A has a melting peak curve with a Tm value of 52 ℃;
judging the first fluorescent channel of the B tube to be positive by HPV18 if the first fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 67 ℃, judging the first fluorescent channel of the B tube to be positive by HPV33 if the first fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 54 ℃, judging the second fluorescent channel of the B tube to be positive by HPV52 if the second fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 69 ℃, judging the second fluorescent channel of the B tube to be positive by HPV43 if the second fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 56 ℃, judging the third fluorescent channel of the B tube to be positive by HPV53 if the third fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 63 ℃, judging the third fluorescent channel of the B tube to be positive by HPV11 if the third fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 51 ℃, judging the fourth fluorescent channel of the B tube to be positive by HPV81, and judging the fourth fluorescent channel of the B tube to be positive by internal quality control;
judging that the HPV18 is positive if the melting peak curve corresponding to the Tm value of 67 ℃ exists in the first fluorescent channel of the C tube, judging that the HPV35 is positive if the melting peak curve corresponding to the Tm value of 56 ℃ exists in the first fluorescent channel of the C tube, judging that the HPV52 is positive if the melting peak curve corresponding to the Tm value of 69 ℃ exists in the second fluorescent channel of the C tube, judging that the HPV44 is positive if the melting peak curve corresponding to the Tm value of 54 ℃ exists in the second fluorescent channel of the C tube, judging that the HPV56 is positive if the melting peak curve corresponding to the Tm value of 62 ℃ exists in the third fluorescent channel of the C tube, judging that the HPV66 is positive if the melting peak curve corresponding to the Tm value of 53 ℃ exists in the third fluorescent channel of the C tube, judging that the sample is PV82 positive if the melting peak curve corresponding to the Tm value of 61 ℃ exists in the fourth fluorescent channel of the C tube, and judging that the internal quality control is qualified if the melting peak curve corresponding to the Tm value of 52 ℃ exists in the fourth fluorescent channel of the C tube;
if the D tube first fluorescence channel has a melting peak curve with a Tm value of 66 ℃, the D tube first fluorescence channel is judged to be HPV16 positive, if the D tube first fluorescence channel has a melting peak curve with a Tm value of 57 ℃, the D tube second fluorescence channel has a melting peak curve with a Tm value of 65 ℃, the D tube second fluorescence channel is judged to be HPV58 positive, if the D tube second fluorescence channel has a melting peak curve with a Tm value of 55 ℃, the D tube second fluorescence channel is judged to be HPV45 positive, if the D tube third fluorescence channel has a melting peak curve with a Tm value of 60 ℃, the D tube third fluorescence channel is judged to be HPV59 positive, if the D tube third fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D tube third fluorescence channel is judged to be HPV68 positive, if the D tube fourth fluorescence channel has a melting peak curve with a Tm value of 62 ℃, the D tube fourth fluorescence channel is judged to be HPV83 positive, and if the D tube fourth fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D tube fourth fluorescence channel is judged to be positive internal quality control qualified.
Wherein, the sample to be detected can be a cervical swab sample of a patient, and the PCR amplification conditions can be as follows: 98 ℃,60 s, (98 ℃, 5s, 58 ℃, 5s, 72 ℃, 5s, 49 cycles); dissolution curve analysis: the temperature is 98 ℃,60 s, 35 ℃,60 s, and the reduction rate is 1.0 ℃/s; fluorescence was collected at 80 ℃ for 5s with a liter of 0.5 ℃/s.
The method disclosed by the invention can sensitively and specifically realize the systematic screening of the human papillomaviruses including HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type within 80 minutes, has simple detection flow, automatically judges and is reliable, and saves time, labor and reagent cost.
The present disclosure is further illustrated in detail below by way of examples, but the present disclosure is not limited thereto.
In the following examples, the reagents were all commercially available, and the primers and probes were synthesized by Biosearch (USA).
Examples
1. Primer and probe synthesis
Sequence synthesis was performed according to the primer sequences shown in Table 1 and the probe sequences shown in Table 2. Y in the sequence represents degenerate base T/C; r represents a degenerate base A/G; w represents a degenerate base A/T; in the probe, FAM is 6-carboxyfluorescein, JOE is 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein, TAMRA is 6-carboxytetramethylrhodamine, and CY5 is 5H-indocyanine. The parenthesis in the probe sequences of Table 2 indicates that t on the left side of the parenthesis has a fluorescent label, and the content in the parenthesis indicates the selection of the fluorescent label.
TABLE 1
TABLE 2
2. Sample processing
After a cervical swab sample of a patient is collected by a conventional method, cervical exfoliated cells on the swab are collected by using a sampler matched with ParaDNA, and the cervical exfoliated cells are directly placed in a ParaDNA reactor for amplification.
3. Detection system for constructing Hybeacon probe technology
Polymerase Phire Hot Start II DNA Polymerase (cat # F122L), Mg2+The dNTPS is purchased from ThermoFisher company, and other biochemical reagents are imported separately packaged or domestic analytical purifiers; the fluorescence detector is ParaDNA.
The reaction system was prepared as follows:
the reaction system was prepared as follows: total system 30. mu.L. 15 mu L of 2 XPCR Buffer, 3-4mM of magnesium chloride solution, 1-1.5 mM of dNTPS, 0.5-1.0 mu M of upstream primer, 0.1-0.5 mu M of downstream primer, 0.1-0.3 mu M of Hybeacon probe, 1.2 mu L of polymerase and 5 mu L of template, wherein the specific primer and probe contents are shown in Table 3, and the rest is complemented with water.
TABLE 3
The kit is divided into 4 reaction tubes of tube A, tube B, tube C and tube D, wherein the tube A contains primers shown by SEQ ID NO.1-2,5-6,9-12,19-20,27-28,43-44 and 51-52 in the table 1 above and probes shown by SEQ ID NO.53,55,57-58,62,66,74 and 78 in the table 2 above; tube B contains the primers shown in SEQ ID Nos. 3-4,7-8,13-14,21-22,29-32,45-46,51-52 of Table 1 above and the probes shown in SEQ ID Nos. 54,56,59,63,67,68,75,78 of Table 2 above; the C-channel contains the primers shown by SEQ ID NO.7-8,15-16,23-24,29-30,33-34,39-40,47-48,51-52 in Table 1 above and the probes shown by SEQ ID NO.56,60,64,67,69,72,76,78 in Table 2 above; tube D contained the primers shown in SEQ ID Nos. 5-6,17-18,25-26,35-38,41-42,49-50,51-52 of Table 1 above and the probes shown in SEQ ID Nos. 55,61,65,70,71,73,77,78 of Table 2 above.
Reaction conditions are as follows: FAM, JOE, CY5 and TAMRA were chosen as reporter groups and the reaction procedure was as follows: 98 ℃,60 s, (98 ℃, 10s, 58 ℃, 10s, 30-40 cycles); dissolution curve analysis: the temperature is 98 ℃,60 s, 35 ℃,60 s, and the reduction rate is 1.0 ℃/s; fluorescence was collected at 80 ℃ for 5s with a liter of 0.5 ℃/s.
And (3) judging a reaction result:
if the positive control and the negative control are established, the detection result is valid;
judging that the HPV16 is positive if the melting peak curve corresponding to the Tm value of 66 ℃ is in the A tube FAM fluorescent channel, judging that the HPV31 is positive if the melting peak curve corresponding to the Tm value of 58 ℃ is in the A tube FAM fluorescent channel, judging that the HPV26 is positive if the melting peak curve corresponding to the Tm value of 65 ℃ is in the A tube JOE fluorescent channel, judging that the HPV42 is positive if the melting peak curve corresponding to the Tm value of 55 ℃ is in the A tube JOE fluorescent channel, judging that the HPV51 is positive if the melting peak curve corresponding to the Tm value of 68 ℃ is in the A tube TAMRA fluorescent channel, judging that the HPV6 is positive if the melting peak curve corresponding to the Tm value of 56 ℃ is in the A tube TAMRA fluorescent channel, judging that the HPV73 is positive if the melting peak curve corresponding to the Tm value of 63 ℃ is in the A tube CY5 fluorescent channel, and judging that the internal quality control is qualified if the melting peak curve corresponding to the Tm value of 52 ℃ is in the A tube CY5 fluorescent channel;
judging that the HPV18 is positive if a melting peak curve corresponding to the Tm value of 67 ℃ exists in a B-tube FAM fluorescent channel, judging that the HPV33 is positive if a melting peak curve corresponding to the Tm value of 54 ℃ exists in the B-tube FAM fluorescent channel, judging that the HPV52 is positive if a melting peak curve corresponding to the Tm value of 69 ℃ exists in a B-tube JOE fluorescent channel, judging that the HPV43 is positive if a melting peak curve corresponding to the Tm value of 56 ℃ exists in the B-tube JOE fluorescent channel, judging that the HPV53 is positive if a melting peak curve corresponding to the Tm value of 63 ℃ exists in a B-tube TAMRA fluorescent channel, judging that the HPV11 is positive if a melting peak curve corresponding to the Tm value of 51 ℃ exists in the B-tube TAMRA fluorescent channel, judging that the HPV81 is positive if a melting peak curve corresponding to the Tm value of 60 ℃ exists in a B-tube CY5 fluorescent channel, and judging that the internal quality control is qualified if a melting peak curve corresponding to the Tm value of 52 ℃ exists in a B-tube CY5 fluorescent channel;
judging the test result to be positive by HPV18 if the C-tube FAM fluorescent channel has a melting peak curve with a Tm value of 67 ℃, judging the test result to be positive by HPV35 if the C-tube FAM fluorescent channel has a melting peak curve with a Tm value of 56 ℃, judging the test result to be positive by HPV52 if the C-tube JOE fluorescent channel has a melting peak curve with a Tm value of 69 ℃, judging the test result to be positive by HPV44 if the C-tube JOE fluorescent channel has a melting peak curve with a Tm value of 54 ℃, judging the test result to be positive by HPV56 if the C-tube TAMRA fluorescent channel has a melting peak curve with a Tm value of 62 ℃, judging the test result to be positive by HPV66 if the C-tube TAMRA fluorescent channel has a melting peak curve with a Tm value of 53 ℃, judging the test result to be positive by PV82 if the C-tube CY5 fluorescent channel has a melting peak curve with a Tm value of 61 ℃, and judging the test result to be positive internal quality control qualified if the C-tube CY5 fluorescent channel has a melting peak curve with a Tm value of 52 ℃;
if the D-tube FAM fluorescence channel has a melting peak curve with a Tm value of 66 ℃, the D-tube FAM fluorescence channel is judged to be HPV16 positive, if the D-tube FAM fluorescence channel has a melting peak curve with a Tm value of 57 ℃, the D-tube JOE fluorescence channel has a melting peak curve with a Tm value of 65 ℃, the D-tube JOE fluorescence channel is judged to be HPV58 positive, if the D-tube JOE fluorescence channel has a melting peak curve with a Tm value of 55 ℃, the D-tube JOE fluorescence channel is judged to be HPV45 positive, if the D-tube TAMRA fluorescence channel has a melting peak curve with a Tm value of 60 ℃, the D-tube TAMRA fluorescence channel is judged to be HPV59 positive, if the D-tube TAMRA fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D-tube CY5 fluorescence channel has a melting peak curve with a Tm value of 62 ℃, the D-tube CY5 fluorescence channel has a melting peak curve with a Tm value of 52 ℃, and the D-tube CY5 fluorescence channel is judged to be HPV68 positive quality control qualified.
4. Specificity verification
Clinical samples (all the samples are from national CDC) such as human papilloma virus HPV 40 type, HPV54 type, HPV61 type, HPV67 type, HPV69 type, HPV70 type, HPV71 type, HPV72 type, herpes simplex virus II type, treponema pallidum, ureaplasma urealyticum, mycoplasma hominis, Neisseria gonorrhoeae, Candida albicans, Trichomonas vaginalis, Chlamydia trachomatis, Acinetobacter baumannii, enterococcus faecalis and the like are selected as specificity evaluation samples, and after a sampler in system detection is adopted to collect cervical exfoliated cells, the cervical exfoliated cells are detected on ParaDNA by utilizing the reaction conditions established and optimized in the early stage.
The result shows that under the condition that positive control is established, the target to be detected has no specific dissolution peak, and the nucleic acid reagent disclosed by the invention can effectively distinguish the detected target from the non-detected target and has better specificity.
5. Minimum detection limit verification
Test samples for evaluation: selecting the initial concentration to be 105The nucleic acid gradient dilution of copies/. mu.L of human papillomavirus HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type is 104Copy/. mu.L, 103Copy/. mu.L, 102Copy/. mu.L, 10 copies/. mu.L, 100Copy/. mu.L, as template for lowest detection limit evaluation. The test was carried out according to the above-mentioned reaction system and reaction procedure.
The results show that the lowest detection limit of the kit of the present disclosure can reach 10 copies/reaction.
6. Coverage verification
Cervical swab samples were selected as templates for coverage assessment. The test was carried out according to the above-mentioned reaction system and reaction procedure.
The results showed that the detection was coverable for all 250 samples.
7. Shelf life test of kit
Respectively taking strong positive 105CFU/mL and Weak Positive 103CFU/mL of human papillomavirus HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HP typeNucleic acid templates of V43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type, which are used as test samples for evaluation, were divided into 10 portions and frozen in a-70 ℃ refrigerator on day 0. And (3) storing the assembled kit at the temperature of-20 ℃, and performing storage period tests on the kit with the time periods of 0, 10, 15, 30, 60, 90, 120, 150, 180 and 360 days respectively.
The results show that the kit disclosed by the invention is stored in a refrigerator at the temperature of-20 ℃, and the detection is positive in different storage periods, which indicates that the storage period of the kit is at least one year.
Comparative example
1. Primer and probe synthesis
Sequence synthesis was performed according to the primer and probe sequences shown in tables 4 and 5. Y in the sequence represents degenerate base T/C; r represents a degenerate base A/G; w represents a degenerate base A/T; in the probe, FAM is 6-carboxyfluorescein, JOE is 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein, TAMRA is 6-carboxytetramethylrhodamine, and CY5 is 5H-indocyanine. The parenthesis in the probe sequences of Table 5 indicates that t to the left of the parenthesis has a fluorescent label, and the content in the parenthesis indicates the selection of the fluorescent label.
TABLE 4
TABLE 5
2. Specificity verification
Specificity verification was performed according to the method of the examples. The results showed that the reaction results of the primers and the probes of the comparative examples were negative.
3. Minimum detection limit verification
The minimum detection limit verification was performed according to the method of the example. The lowest detection limit of the examples versus the comparative examples is shown in table 6 below.
TABLE 6
| Detecting an object | Examples | Comparative example |
| HPV6 | 10 copies/reaction | 100 copies/reaction |
| HPV11 | 10 copies/reaction | 100 copies/reaction |
| HPV16 | 10 copies/reaction | 100 copies/reaction |
| HPV18 | 10 copies/reaction | 50 copies/reaction |
| HPV26 | 10 copies/reaction | 100 copies/reaction |
| HPV31 | 10 copies/reaction | 100 copies/reaction |
| HPV33 | 10 copies/reaction | 100 copies/reaction |
| HPV35 | 10 copies/reaction | 50 copies/reaction |
| HPV39 | 10 copies/reaction | 100 copies/reaction |
| HPV42 | 10 copies/reaction | 50 copies/reaction |
| HPV43 | 10 copies/reaction | 100 copies/reaction |
| HPV44 | 10 copies/reaction | 50 copies/reaction |
| HPV45 | 10 copies/reaction | 100 copies/reaction |
| HPV51 | 10 copies/reaction | 100 copies/reaction |
| HPV52 | 10 copies/reaction | 50 copies/reaction |
| HPV53 | 10 copies/reaction | 100 copies/reaction |
| HPV56 | 10 copies/reaction | 100 copies/reaction |
| HPV58 | 10 copies/reaction | 50 copies/reaction |
| HPV59 | 10 copies/reaction | 100 copies/reaction |
| HPV66 | 10 copies/reaction | 100 copies/reaction |
| HPV68 | 10 copies/reaction | 100 copies/reaction |
| HPV73 | 10 copies/reaction | 50 copies/reaction |
| HPV81 | 10 copies/reaction | 100 copies/reaction |
| HPV82 | 10 copies/reaction | 100 copies/reaction |
| HPV83 | 10 copies/reaction | 100 copies/reaction |
As can be seen from Table 6, the disclosed kit has a stronger detection capability than the comparative examples for trace amounts of human papillomavirus HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type nucleic acids in a sample.
4. Coverage verification
Coverage verification was performed as per the method of the examples. The coverage ratio of examples to comparative examples is shown in table 7 below.
TABLE 7
As can be seen from Table 7, the detection coverage of the kit of the present disclosure is much greater than that of the comparative example.
As can be seen from comparison of examples and comparative examples, the present disclosure can detect human papillomaviruses including HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type and HPV83 type at a time, and has high specificity, lower minimum detection limit and wider coverage.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Sequence listing
<110> Beijing Zhuozcheng Biotech GmbH
<120> nucleic acid reagents, kits, systems and methods for detecting human papillomavirus
<130> 12325ABT
<160> 156
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggacggacaa gattcacaac ctttaaa 27
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtacacccag acccctcatt tt 22
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cctttaacac aacattacca aata 24
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tctactatgg cttctaccat aaa 23
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgagcaatta aatgacagct cagag 25
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgagaacaga tggggcacac aat 23
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaccttctat gtcacgagca atta 24
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tgcacaccac ggacacacaa ag 22
<210> 9
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cgaaattgac ctacgctgct acg 23
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tggcacacca aggacacgtc ttc 23
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
agcaattacc cgacagctca gat 23
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gtagaacagt tggggcacac ga 22
<210> 13
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
actgacctay actgctatga gcaa 24
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tgtgcacags tagggcacac aat 23
<210> 15
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
caactgacct atactgttat gagc 24
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
tgtgaacagc cggggcacac ta 22
<210> 17
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ttgtatgtca cgagcaatta ggag 24
<210> 18
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gacactgtgt cgcctgtttg ttta 24
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
cagatgaaga tgaccaagcc aaa 23
<210> 20
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ttgtctacta tagcttctac acaaaa 26
<210> 21
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ccagcaagtg aatctacaag ttta 24
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gtccactgtt gcctctacta taaa 24
<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gacgttacac agccttacca aata 24
<210> 24
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tctccactat agcctctact agaaa 25
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gacctgttgt gttacgagca atta 24
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tgcacaccac ggacacacaa ag 22
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gctacgagca atttgacagc tcag 24
<210> 28
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
atcgccgttg ctagttgttc gca 23
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
actgacctay actgctatga gcaa 24
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cagccggggc acacaacttg taa 23
<210> 31
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
acctgcaatg ccatgagcaa ttgaa 25
<210> 32
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
ttatcgcctt gttgcgcaga gg 22
<210> 33
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
acctacartg caatgagcaa ttgg 24
<210> 34
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
tgatgcgcag agtgggcacg tta 23
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gctatgagca attatgtgac agct 24
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tgtgcacags tagggcacac aat 23
<210> 37
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
accttgtgtg ctacgagcaa ttac 24
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gctgcacaca aaggacacac aaa 23
<210> 39
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
acctacartg caatgagcaa ttgg 24
<210> 40
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tgatgcgcag agtgggcacg tta 23
<210> 41
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ttgtatgtca cgagcaatta ggag 24
<210> 42
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gattactggg tttccgttgc acac 24
<210> 43
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
cttacatgtt acgagtcatt ggac 24
<210> 44
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gtttctggaa cagttggggc ac 22
<210> 45
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gtttagttag gctggtggtg ttaa 24
<210> 46
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ctacaacagc ctccaccata aa 22
<210> 47
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gctacgagca atttgacagc tcag 24
<210> 48
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
cattgccgat gttagttggt cgca 24
<210> 49
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ggatttggaa gacttacgcc ttattt 26
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
ctctcgcacc tttgctgctt t 21
<210> 51
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tttggacctg cgagcg 16
<210> 52
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gagcggctgt ctccacaagt 20
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
aacgttcgac tggttgtgca 20
<210> 54
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
atgtgacagc aacgtccgac tg 22
<210> 55
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
tagatggtcc agctggacaa g 21
<210> 56
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
tagatggagt taatcatcaa ca 22
<210> 57
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
agatgaagga tgaaacagat aatatg 26
<210> 58
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
acagtccagc tggacaagca g 21
<210> 59
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
ccagatgagg atgaaggctt gga 23
<210> 60
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
attgacggtc cagctggaca a 21
<210> 61
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ggagagtcag aggatgaaat a 21
<210> 62
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
aagtctgtta aactcgttgt gc 22
<210> 63
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
agtgctctga cagtgacatc a 21
<210> 64
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
aaggttcggc tggttgtgca g 21
<210> 65
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
atgaagcaga tggagttagt c 21
<210> 66
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ggctggacag gctacgtgtt a 21
<210> 67
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
attgtgacat attgtcacag t 21
<210> 68
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
atgaggatga agtagaccat 20
<210> 69
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
aggatgagga tgaggatgaa gta 23
<210> 70
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
attgtaactt gttgttacac t 21
<210> 71
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
attgtgtgtg tgtgttgtaa g 21
<210> 72
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
atgaggatga aatagaccat ttgc 24
<210> 73
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
cagtgtacgt gttgtaagtg taa 23
<210> 74
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
agttactgac tgcacgaagt 20
<210> 75
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
agtgtgtcct ggctgcgcat 20
<210> 76
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
agtagataat atgcgtgacc a 21
<210> 77
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
catggaagga gctgtgtatt aa 22
<210> 78
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gttctgacct gaaggctct 19
<210> 79
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
cacaaccttt aaaacaacat ttccaaata 29
<210> 80
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
tcgtccgcca tcgttgttat 20
<210> 81
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
gacatcagac aactacaaga cctttt 26
<210> 82
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
caccccgacc cctcatttt 19
<210> 83
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
gagcccatta caatattgta acctttt 27
<210> 84
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
gaacagatgg ggcacacaat t 21
<210> 85
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
gagcaattaa gcgactcaga ggaa 24
<210> 86
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gaaggtcgtc tgctgagctt t 21
<210> 87
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gccagacaag ctggacaaga a 21
<210> 88
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
caccaaggac acgtcttcca ttaa 24
<210> 89
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
cacatccaat tacaatatcg ttacctttt 29
<210> 90
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
ttggggcaca cgattccaaa t 21
<210> 91
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
cacttgtaac accacagttc gtttat 26
<210> 92
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
gatcggccat tgtagatgat gttta 25
<210> 93
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
aaccagacac ctccaattat aat 23
<210> 94
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
attccaaatg tgcccattaa taaat 25
<210> 95
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
gaacccgacc atgcagttaa t 21
<210> 96
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
acacacaaat cctagtgagt ccataaa 27
<210> 97
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
gaaacctgca acagatgctt tt 22
<210> 98
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
tttgtctact atagcttcta cacaaaa 27
<210> 99
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
gacctgctgt tgggcacatt aaa 23
<210> 100
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
gtcccactgt tgcctctact ataaa 25
<210> 101
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
ctgctgggtt cactggatat att 23
<210> 102
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
gttctccact atagcctcta ctagaaa 27
<210> 103
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
cgaaccacag cgtcacaaaa tt 22
<210> 104
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
caaaggacaa ggtgctcaaa aa 22
<210> 105
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
gcaggtgttc aagtgtagta caa 23
<210> 106
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
ttcgcacaac acgggcaaa 19
<210> 107
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
gcagaacaag ccacaagcaa tta 23
<210> 108
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
ccggggcaca caacttgtaa t 21
<210> 109
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
gggacgaaca acatccttgt t 21
<210> 110
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
tgcccataag catttgttgt aaaat 25
<210> 111
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
gtgttaccta atacacgtac cttgtt 26
<210> 112
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
agagtgggca cgttactgtt aa 22
<210> 113
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
tggcaccacg gttcgttt 18
<210> 114
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
catccattgc agatggtgtt tatt 24
<210> 115
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
gagccttaca gcagctgttt at 22
<210> 116
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
gtaccttccg aatcggccat t 21
<210> 117
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
ttacctaatt cacgtacctt gttgtaa 27
<210> 118
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
ggtgatgcca ttgcagttat tt 22
<210> 119
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
ctgctgttta tggactcact aaattt 26
<210> 120
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
tcatcaatgt cattggccat gtt 23
<210> 121
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
ccttgccatt gaaagcaaca aa 22
<210> 122
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
acctgaatca gccatcttct ttt 23
<210> 123
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
cctgaggctg ctgcaacaa 19
<210> 124
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
ctacaacagc ctccaccata aa 22
<210> 125
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
ggacaggata cgtgttacag aattaaa 27
<210> 126
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
cttaggtcgc ccagtaacat tt 22
<210> 127
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
cggtacccca ccaacatatt t 21
<210> 128
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
gcagttcgtg gtcccactta ata 23
<210> 129
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
atggcggtgt ttgcagattt 20
<210> 130
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
gattgatagc aacaactgaa tagccaa 27
<210> 131
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
actggttgtg cagtgtac 18
<210> 132
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
tgtgtcccat ctgcgcac 18
<210> 133
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
cggttgtgcg tacaaagcac a 21
<210> 134
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
tgttgtgtat gtgttgtaag t 21
<210> 135
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
agtatagtgc agctagcag 19
<210> 136
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
tcagtgtaag tctacacttc g 21
<210> 137
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
agtgaatatt gtgtgcccta c 21
<210> 138
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
tgtaaatgtg aggcgacact a 21
<210> 139
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
acagtgttcg tgttgtaagt aa 22
<210> 140
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
gagtaactgc aatggcggat g 21
<210> 141
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
ggtgtgacta ccatggctga taa 23
<210> 142
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
atggctgaca atacaggtac ag 22
<210> 143
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
attgagctta cagtagagag c 21
<210> 144
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
gcgttgtaca gcagatgtta a 21
<210> 145
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
ttgtcacagt tgtgatagca ca 22
<210> 146
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
tgtaggtgtg agtcgttggt gc 22
<210> 147
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
taagtttgtg gtgcagttgg a 21
<210> 148
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
agtacaacaa ccgacgtacg a 21
<210> 149
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
tgtgtgcagc aaaccagtaa cct 23
<210> 150
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
agttggtggt gcagttggac a 21
<210> 151
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
atggccaatt gtgaagggct c 21
<210> 152
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
agttgcttat gggtacacta g 21
<210> 153
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
agtgacatgg cagatgtgga a 21
<210> 154
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
agtgttgtac agctcgcagt g 21
<210> 155
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
gaggtggatt tggaagactt a 21
<210> 156
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
cctgaaggct ctgcgcggac t 21
Claims (8)
1. A nucleic acid reagent for detecting human papilloma virus, wherein the nucleic acid reagent comprises primers shown in SEQ ID NO.1-50 and Hybeacon probes shown in SEQ ID NO. 53-77;
the nucleic acid reagent also comprises a positive internal quality control;
the positive endoplasmic control contains a primer shown by SEQ ID NO.51-52 and a Hybeacon probe shown by SEQ ID NO. 78;
the nucleic acid reagent comprises primers shown in SEQ ID NO.1-52 and Hybeacon probe shown in SEQ ID NO.53-78 which are independently stored, or
The nucleic acid reagent comprises a tube A, a tube B, a tube C and a tube D; the tube A contains primers shown in SEQ ID NO.1-2,5-6,9-12,19-20,27-28,43-44 and 51-52 and Hybeacon probes shown in SEQ ID NO.53,55,57-58,62,66,74 and 78; the tube B contains primers shown in SEQ ID NO.3-4,7-8,13-14,21-22,29-32,45-46 and 51-52 and Hybeacon probes shown in SEQ ID NO.54,56,59,63,67,68,75 and 78; the C tube contains primers shown in SEQ ID NO.7-8,15-16,23-24,29-30,33-34,39-40,47-48 and 51-52 and Hybeacon probes shown in SEQ ID NO.56,60,64,67,69,72,76 and 78; the D tube contains primers shown in SEQ ID NO.5-6,17-18,25-26,35-38,41-42,49-50 and 51-52 and Hybeacon probes shown in SEQ ID NO.55,61,65,70,71,73,77 and 78.
2. The nucleic acid reagent according to claim 1, wherein the content of the primer represented by SEQ ID NO.2-50 is 0.1-0.5. mu.M, 0.5-1.0. mu.M, 0.1-0.5. mu.M, 0.5-1.0. mu.0. mu.M, 0.1-0.5. mu.M, 0.5-1.0.5. mu.0.5. mu.M, 0.5-1-0.5. mu.0.5. mu.M, 0.5. mu.0.M, 0.5. mu.5-1-0.5. mu.0.5. mu.0.M, 0.5. mu.5. mu.0.5. mu.5. mu.M, 0.0.5. mu.5-1-0.0.5. mu.0.M, 0.5. mu.5. mu.0.M, 0.0.5. mu.5. mu.0.0.0.5. mu.M, 0.5. mu.0.5. mu.5 to 0.5. mu.0.5. mu.M, 0.0.M, 0.5 to 0.5. mu.0.M, 0.0.0.0.5. mu.0.5 to 0.0.5. mu.5 to 0.5. mu.M, 0.5. mu.0.0.5 to 0.0.0.M, 0.5 to 0.M, 0.5. mu.0.0.0.0.0.0.0.0.0.0.0.5 to 1 to 1.5. mu.M, 0.5 to 0.0.0.5 to 1.0.M, 0.0.0.M, 0.5 to 0.0.0.5 to 1.0.0.0.5. mu.5 to 0.5. mu.5. mu.M, 0.0.5 to 0.0.M, 0.0.0.0.0.0.0.0.0.M, 0.5 to 0.0.0.5 to 0.M, 0.0.0.5 to 0.0.M, 0.0.0.0.5 to 1.M, 0.0.M, 0.5 to 0.0.0.0.0.0.0.0.0.0.0.0.5 to 1.0.5 to 1 to 0.0.5 to 1.5 to 1 to 1.5 to 1.M, 0.5 to 1 to 1.5 to 0.0.5 to 1 to 0.5 to 0.0.5 to 0.5 to 0.M, 0.5 to 0.0.5 to 0.0.M, 0.5 to 0.0.0.0.0.5 to 1 to 1.0.5 to 1 to, 0.5 to 1.0. mu.M, 0.1 to 0.5. mu.M, 0.5 to 1.0. mu.M and 0.1 to 0.5. mu.M, and the content of the Hyberacon probe represented by SEQ ID Nos. 53 to 77 is 0.1 to 0.3. mu.M each independently.
3. The nucleic acid reagent according to claim 1, wherein the Hybeacon probe shown in SEQ ID nos. 55-56,58-61 has a first fluorescent label; the Hybeacon probe shown in SEQ ID NO.57,62-65,67,70 has a second fluorescent label; the Hybeacon probe shown in SEQ ID NO.53,54,66,68-69,71-73 has a third fluorescent label; the Hybeacon probe shown in SEQ ID NO.74-78 has a fourth fluorescent label; the first fluorescent label, the second fluorescent label, the third fluorescent label and the fourth fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a JOE fluorescent label, a TAMRA fluorescent label, a CY5 fluorescent label, a ROX fluorescent label and a Quasar670 fluorescent label.
4. The nucleic acid reagent according to any one of claims 1 to 3, wherein the human papillomavirus comprises at least one of HPV6 type, HPV11 type, HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV42 type, HPV43 type, HPV44 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV81 type, HPV82 type, and HPV83 type.
5. A kit for detecting human papillomavirus, the kit comprising the nucleic acid reagent of any of claims 1 to 4.
6. The kit of claim 5, wherein the kit further comprises at least one of a reaction system buffer, a DNA polymerase, magnesium ions, dNTPs, and water.
7. Use of the nucleic acid reagent of any one of claims 1-4 in the preparation of a kit for detecting human papillomavirus.
8. A system for detecting human papillomavirus, the system comprising a PCR instrument having an a-tube detector, a B-tube detector, a C-tube detector and a D-tube detector, the a-tube detector, the B-tube detector, the C-tube detector and the D-tube detector being respectively nucleic acid reagent storage containers loaded with the nucleic acid reagents of any one of claims 4 to 6, the PCR instrument comprising a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel, the first fluorescence channel, the second fluorescence channel, the third fluorescence channel and the fourth fluorescence channel being different and each independently being a FAM fluorescence channel, a JOE fluorescence channel, a TAMRA fluorescence channel, a CY5 fluorescence channel, a ROX fluorescence channel or a Quasar670 fluorescence channel; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
if the positive control and the negative control are established, the detection result is valid;
judging the first fluorescent channel of the tube A to be positive by HPV16 if the first fluorescent channel of the tube A has a melting peak curve with a Tm value of 66 ℃, judging the first fluorescent channel of the tube A to be positive by HPV31 if the first fluorescent channel of the tube A has a melting peak curve with a Tm value of 58 ℃, judging the second fluorescent channel of the tube A to be positive by HPV26 if the second fluorescent channel of the tube A has a melting peak curve with a Tm value of 65 ℃, judging the second fluorescent channel of the tube A to be positive by HPV42 if the second fluorescent channel of the tube A has a melting peak curve with a Tm value of 55 ℃, judging the third fluorescent channel of the tube A to be positive by HPV51 if the third fluorescent channel of the tube A has a melting peak curve with a Tm value of 56 ℃, judging the third fluorescent channel of the tube A to be positive by HPV6 if the fourth fluorescent channel of the tube A has a melting peak curve with a Tm value of 63 ℃, and judging the fourth fluorescent channel of the tube A to be qualified for internal quality control if the fourth fluorescent channel of the tube A has a melting peak curve with a Tm value of 52 ℃;
judging the first fluorescent channel of the B tube to be positive by HPV18 if the first fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 67 ℃, judging the first fluorescent channel of the B tube to be positive by HPV33 if the first fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 54 ℃, judging the second fluorescent channel of the B tube to be positive by HPV52 if the second fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 69 ℃, judging the second fluorescent channel of the B tube to be positive by HPV43 if the second fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 56 ℃, judging the third fluorescent channel of the B tube to be positive by HPV53 if the third fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 63 ℃, judging the third fluorescent channel of the B tube to be positive by HPV11 if the third fluorescent channel of the B tube has a melting peak curve corresponding to the Tm value of 51 ℃, judging the fourth fluorescent channel of the B tube to be positive by HPV81, and judging the fourth fluorescent channel of the B tube to be positive by internal quality control;
judging that the HPV18 is positive if the melting peak curve corresponding to the Tm value of 67 ℃ exists in the first fluorescent channel of the C tube, judging that the HPV35 is positive if the melting peak curve corresponding to the Tm value of 56 ℃ exists in the first fluorescent channel of the C tube, judging that the HPV52 is positive if the melting peak curve corresponding to the Tm value of 69 ℃ exists in the second fluorescent channel of the C tube, judging that the HPV44 is positive if the melting peak curve corresponding to the Tm value of 54 ℃ exists in the second fluorescent channel of the C tube, judging that the HPV56 is positive if the melting peak curve corresponding to the Tm value of 62 ℃ exists in the third fluorescent channel of the C tube, judging that the HPV66 is positive if the melting peak curve corresponding to the Tm value of 53 ℃ exists in the third fluorescent channel of the C tube, judging that the sample is positive by PV82 if the melting peak curve corresponding to the Tm value of 61 ℃ exists in the fourth fluorescent channel of the C tube, and judging that the internal quality control is qualified if the melting peak curve corresponding to the Tm value of 52 ℃ exists in the fourth fluorescent channel of the C tube;
if the D tube first fluorescence channel has a melting peak curve with a Tm value of 66 ℃, the D tube first fluorescence channel is judged to be HPV16 positive, if the D tube first fluorescence channel has a melting peak curve with a Tm value of 57 ℃, the D tube second fluorescence channel has a melting peak curve with a Tm value of 65 ℃, the D tube second fluorescence channel is judged to be HPV58 positive, if the D tube second fluorescence channel has a melting peak curve with a Tm value of 55 ℃, the D tube second fluorescence channel is judged to be HPV45 positive, if the D tube third fluorescence channel has a melting peak curve with a Tm value of 60 ℃, the D tube third fluorescence channel is judged to be HPV59 positive, if the D tube third fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D tube third fluorescence channel is judged to be HPV68 positive, if the D tube fourth fluorescence channel has a melting peak curve with a Tm value of 62 ℃, the D tube fourth fluorescence channel is judged to be HPV83 positive, and if the D tube fourth fluorescence channel has a melting peak curve with a Tm value of 52 ℃, the D tube fourth fluorescence channel is judged to be positive internal quality control qualified.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811641953.0A CN109609696B (en) | 2018-12-29 | 2018-12-29 | Nucleic acid reagent, kit, system and method for detecting human papilloma virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811641953.0A CN109609696B (en) | 2018-12-29 | 2018-12-29 | Nucleic acid reagent, kit, system and method for detecting human papilloma virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109609696A CN109609696A (en) | 2019-04-12 |
| CN109609696B true CN109609696B (en) | 2021-10-01 |
Family
ID=66017395
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811641953.0A Active CN109609696B (en) | 2018-12-29 | 2018-12-29 | Nucleic acid reagent, kit, system and method for detecting human papilloma virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109609696B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819739A (en) * | 2019-11-29 | 2020-02-21 | 广东龙帆生物科技有限公司 | Saffold virus real-time fluorescent PCR detection primer, probe, detection kit, detection method and application |
| CN113584225B (en) * | 2021-07-27 | 2022-06-28 | 赵飞 | Primer and probe combination for detecting HPV (human papillomavirus) virus, typing detection reagent and application thereof |
| CN116287442A (en) * | 2022-12-23 | 2023-06-23 | 上海宝藤生物医药科技股份有限公司 | Primer probe combination for detecting human papilloma virus and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1948503A (en) * | 2005-10-10 | 2007-04-18 | 上海透景生命科技有限公司 | Detection and parting method of human papillomavirus and reagent box |
| CN101851630A (en) * | 2009-03-30 | 2010-10-06 | 白向阳 | Human papillomavirus HPV DNA fragment, specific primer and application thereof |
| CN106048081A (en) * | 2016-05-19 | 2016-10-26 | 胤安国际（辽宁）基因科技股份有限公司 | HPV (human papilloma virus) typing detection primers as well as detection method and application thereof |
| CN106048080A (en) * | 2016-05-19 | 2016-10-26 | 胤安国际（辽宁）基因科技股份有限公司 | Primer and method for rapidly screening HPV (human papillomavirus) sub-types |
-
2018
- 2018-12-29 CN CN201811641953.0A patent/CN109609696B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1948503A (en) * | 2005-10-10 | 2007-04-18 | 上海透景生命科技有限公司 | Detection and parting method of human papillomavirus and reagent box |
| CN101851630A (en) * | 2009-03-30 | 2010-10-06 | 白向阳 | Human papillomavirus HPV DNA fragment, specific primer and application thereof |
| CN106048081A (en) * | 2016-05-19 | 2016-10-26 | 胤安国际（辽宁）基因科技股份有限公司 | HPV (human papilloma virus) typing detection primers as well as detection method and application thereof |
| CN106048080A (en) * | 2016-05-19 | 2016-10-26 | 胤安国际（辽宁）基因科技股份有限公司 | Primer and method for rapidly screening HPV (human papillomavirus) sub-types |
Non-Patent Citations (3)
| Title |
|---|
| Combination of fluorescence color and melting temperature as a two-dimensional label for homogeneous multiplex PCR detection;Yiqun Liao等;《Nucleic Acids Research,》;20130118;e76 * |
| HyBeaconTM probes: a new tool for DNA sequence detection and allele discrimination;D. J. French等;《Molecular and Cellular Probes》;20011231;第363–374页 * |
| 高通量荧光PCR 组合方法鉴定16 种金黄色葡萄球菌肠毒素的方法建立及评估;左乐等;《疾病监测》;20180930;第753-757页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109609696A (en) | 2019-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105087827B (en) | Detection primer, probe and the kit of 16 kinds of type HPV viruses | |
| CN101487063B (en) | Human papilloma virus infection gene amplification fluorescent detection kit | |
| CN105755169B (en) | Detection and typing kit for high-risk human papilloma virus and application thereof | |
| CN105603121A (en) | Method, oligonucleotide and kit for detecting high-risk HPV | |
| CN104818342B (en) | Detection kit, detection architecture and method for 19 kinds of high-risk human mammilla papillomavirus (HPV) | |
| CN103725792A (en) | Human papilloma virus (24 types) detection (fluorescent PCR method) kit and detection method | |
| CN109609696B (en) | Nucleic acid reagent, kit, system and method for detecting human papilloma virus | |
| CN116769964A (en) | Primer, primer probe composition and kit for detecting high-risk human papilloma virus | |
| Militello et al. | Accurate human papillomavirus genotyping by 454 pyrosequencing | |
| CN110093459A (en) | For quickly detecting the LAMP primer composition and application of 13 kinds of high-risk HPVs | |
| WO2024187844A1 (en) | Primer and use of composition of same and probe in preparation of kit for detecting hpv | |
| Sait et al. | Genetic diversity and phylogenetic analysis of HPV 16 & 18 variants isolated from cervical specimens of women in Saudi Arabia | |
| CN105803110B (en) | Kit for simultaneously typing and detecting multiple human papilloma viruses and application thereof | |
| CN101886137B (en) | Fluorescent quantitative PCR kit for qualitative detection of high-risk HPV | |
| CN111172328A (en) | Complete set of primers, complete set of probes, kit and method for HPV nucleic acid typing detection | |
| CN104560962B (en) | Fluorescent PCR kit and its primer special group for detecting HPV | |
| CN106048081A (en) | HPV (human papilloma virus) typing detection primers as well as detection method and application thereof | |
| CN108179226B (en) | Nucleic acid composition for detecting human papilloma virus, application thereof and kit | |
| CN119351624A (en) | Primer probe combination, reagent or kit for human papillomavirus nucleic acid detection and application | |
| ES2525233T3 (en) | Identification and quantification of oncogenic HPV nucleic acids through real-time PCR assays | |
| CN102140554B (en) | Fluorescent PCR kit for detecting human papilloma virus subtypes | |
| CN117625844A (en) | Multiplex Q-PCR detection kit for HPV virus and preparation thereof | |
| Dobec et al. | Automation of the linear array HPV genotyping test and its application for routine typing of human papillomaviruses in cervical specimens of women without cytological abnormalities in Switzerland | |
| CN105695627A (en) | Specific primer and probe combination and reagent kit for fluorescent quantitative PCR (polymerase chain reaction) detection on 15 types of HPV (human papillomavirus) | |
| CN111593140B (en) | High-risk human papilloma virus detection and typing kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |