CN117757963A - Primer combination, kit and method for enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection - Google Patents
Primer combination, kit and method for enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection Download PDFInfo
- Publication number
- CN117757963A CN117757963A CN202311809361.6A CN202311809361A CN117757963A CN 117757963 A CN117757963 A CN 117757963A CN 202311809361 A CN202311809361 A CN 202311809361A CN 117757963 A CN117757963 A CN 117757963A
- Authority
- CN
- China
- Prior art keywords
- yersinia enterocolitica
- nucleic acid
- seq
- yersinia
- fluorescence pcr
- 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.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 58
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 41
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 41
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 23
- 241000607734 Yersinia <bacteria> Species 0.000 title claims abstract description 22
- 208000010227 enterocolitis Diseases 0.000 title claims abstract description 20
- 239000000523 sample Substances 0.000 claims abstract description 78
- 241000607447 Yersinia enterocolitica Species 0.000 claims abstract description 67
- 229940098232 yersinia enterocolitica Drugs 0.000 claims abstract description 66
- 230000001018 virulence Effects 0.000 claims abstract description 40
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- 101100125055 Yersinia enterocolitica ystA gene Proteins 0.000 claims abstract description 25
- 101150070603 yadA gene Proteins 0.000 claims abstract description 25
- 101100022900 Shigella flexneri merE gene Proteins 0.000 claims abstract description 24
- 101150099477 ail gene Proteins 0.000 claims abstract description 24
- 101150075391 mtnN gene Proteins 0.000 claims abstract description 24
- 101100125056 Yersinia enterocolitica ystB gene Proteins 0.000 claims abstract description 23
- 101150103224 virF gene Proteins 0.000 claims abstract description 21
- 101100398736 Yersinia pestis lcrF gene Proteins 0.000 claims abstract description 19
- 101150014425 foxA gene Proteins 0.000 claims abstract description 18
- 230000003321 amplification Effects 0.000 claims description 23
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 22
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 239000011541 reaction mixture Substances 0.000 claims description 16
- 238000003908 quality control method Methods 0.000 claims description 13
- 238000000137 annealing Methods 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 7
- 239000013612 plasmid Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000011535 reaction buffer Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- 238000011529 RT qPCR Methods 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 238000012257 pre-denaturation Methods 0.000 claims description 4
- 238000003762 quantitative reverse transcription PCR Methods 0.000 claims description 4
- 102000016911 Deoxyribonucleases Human genes 0.000 claims description 3
- 108010053770 Deoxyribonucleases Proteins 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 238000010257 thawing Methods 0.000 claims description 3
- 235000011178 triphosphate Nutrition 0.000 claims description 3
- 239000001226 triphosphate Substances 0.000 claims description 3
- 102000006382 Ribonucleases Human genes 0.000 claims description 2
- 108010083644 Ribonucleases Proteins 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 239000008213 purified water Substances 0.000 claims description 2
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 claims description 2
- 206010009887 colitis Diseases 0.000 claims 1
- 210000000813 small intestine Anatomy 0.000 claims 1
- 230000007918 pathogenicity Effects 0.000 abstract description 6
- 238000013461 design Methods 0.000 abstract description 4
- 238000011895 specific detection Methods 0.000 abstract 2
- 241000894006 Bacteria Species 0.000 description 7
- 230000001717 pathogenic effect Effects 0.000 description 5
- 101100013363 Yersinia enterocolitica foxA gene Proteins 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 206010012735 Diarrhoea Diseases 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 101150039504 6 gene Proteins 0.000 description 2
- 101710146739 Enterotoxin Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 241000271566 Aves Species 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000589877 Campylobacter coli Species 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241001333951 Escherichia coli O157 Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000244365 Ligusticum sinense Species 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 241000531795 Salmonella enterica subsp. enterica serovar Paratyphi A Species 0.000 description 1
- 241000239226 Scorpiones Species 0.000 description 1
- 241000607762 Shigella flexneri Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 1
- 241000607265 Vibrio vulnificus Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241001377938 Yara Species 0.000 description 1
- 101100108460 Yersinia enterocolitica ail gene Proteins 0.000 description 1
- 101100428579 Yersinia enterocolitica virF gene Proteins 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 201000009840 acute diarrhea Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007847 digital PCR Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 108010037896 heparin-binding hemagglutinin Proteins 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000011022 operating instruction Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 101150017680 ystA gene Proteins 0.000 description 1
- 101150089186 ystB gene Proteins 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer combination, a kit and a detection method for enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection, which are used for judging whether a detected sample contains enterocolitis yersinia and whether the detected sample has pathogenicity according to 6 genes, namely general gene foxA and virulence gene ail, ystA, ystB, yadA, virF of enterocolitis yersinia. The invention designs specific detection primers and probes for the six genes, and designs a multiplex real-time fluorescence PCR detection kit capable of simultaneously detecting the six genes based on the specific detection primers and the probes, thereby realizing the purpose of judging whether the sample to be detected contains the Yersinia enterocolitica and judging whether the sample has pathogenicity. The method has important significance for detecting the yersinia enterocolitica containing different virulence genes.
Description
Technical Field
The invention belongs to the technical field of molecular biology, relates to virus detection, and in particular relates to a primer combination, a kit and a method for enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection.
Background
Yersinia enterocolitica (Y.enterocolitica) is a gram-negative bacterium belonging to the genus Enterobacter, which can cause Yersinia disease. The bacteria are important food-borne pathogenic bacteria, and are zoonotic pathogenic bacteria; the strain has psychrophilic property, can survive and reproduce under the condition of low-temperature refrigeration, and is listed as a conventional detection item for imported and exported foods in many countries.
Yersinia enterocolitica is found worldwide and is widely found in animals such as pigs, cattle, sheep, aquatic products, poultry, birds, insects, and the like. Because of the high bacteria carrying rate of edible animals, the food processing process is also seriously polluted, and the morbidity is mainly related to the intake of polluted food or water. Enterocolitis yersinia can cause clinical diseases such as acute diarrhea, meningitis, myocarditis and the like, and can pose a certain threat to human health, so that attention should be paid to pathogenic bacteria which can be transmitted through food and have psychrophilicity.
Studies have shown that yersinia enterocolitica has a highly conserved gene foxA, which is also an identified gene for yersinia enterocolitica. The virulence of yersinia enterocolitica is regulated by virulence genes on chromosomes and plasmids, and the 5 major virulence genes that are often used for detection are: ail, ystA, ystB, yadA and virF. Wherein ail is used as an adhesion invasion site gene on a chromosome, and is only present in pathogenic strains, and is often used for identifying pathogenicity of the bacteria; the ystA and ystB2 are two subtypes of the yersinia thermo-resistant enterotoxin gene (Yst) on their chromosomes, with ystA being present mainly in pathogenic strains, while ystB is not coexisting with other virulence genes, a thermo-resistant enterotoxin gene of the non-pathogenic organism yersinia enterocolitica type 1A; yadA (adhesin gene A) and virF (virulence activator gene) are virulence genes present on a plasmid.
The current method for detecting the yersinia enterocolitica is mainly based on polymerase chain reaction PCR technology, including real-time fluorescent PCR, digital PCR, isothermal amplification technology and the like, most detection methods can only detect one virulence gene at a time, time consuming and complex to operate, and the multiple fluorescent quantitative PCR method established in recent years is mostly used for detecting serotypes and virulence genotypes, but does not contain yadA and virF virulence genes existing on plasmids, and can not be used for identifying the virulence genes contained in the yersinia enterocolitica at the same time of detecting the yersinia enterocolitica.
Therefore, how to determine whether or not a sample to be tested contains yersinia enterocolitica and to identify whether or not it is pathogenic is still a problem to be solved.
Disclosure of Invention
Aiming at the problem of judging whether the sample contains the yersinia enterocolitica and identifying whether the sample has pathogenicity, the invention provides a primer combination, a kit and a method for detecting the yersinia enterocolitica nucleic acid by multiplex fluorescence PCR, and simultaneously detects foxA, ail, ystA, ystB, yadA, virF 6 genes, and the 6 genes are used for judging whether the sample contains the yersinia enterocolitica and whether the sample has pathogenicity, thereby having great significance for detecting the yersinia enterocolitica containing different virulence genes. The specific technical scheme is as follows:
first, the invention provides a primer combination for enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection, which comprises six primer pairs and six probes for detecting enterocolitis yersinia universal gene foxA and five virulence genes ail, ystA, ystB, yadA, virF genes; the nucleotide sequence of each primer and each probe is shown as SEQ NO. 1-SEQ NO. 18.
A primer combination for the aforementioned yersinia enterocolitica nucleic acid multiplex fluorescent PCR detection, wherein:
the primer pair and the probe shown in SEQ NO. 1-SEQ NO.3 are used for detecting the gene foxA;
the primer pair and the probe shown in SEQ NO. 4-SEQ NO.6 are used for detecting virulence genes ail;
the primer pair and the probe shown in SEQ NO. 7-SEQ NO.9 are used for detecting virulence genes ystA;
the primer pair and the probe shown in SEQ NO. 10-SEQ NO.12 are used for detecting virulence genes ystB;
the primer pair and the probe shown in SEQ NO. 13-SEQ NO.15 are used for detecting virulence genes yadA;
the primer pair and the probe shown in SEQ NO. 16-SEQ NO.18 are used for detecting virulence gene virF.
The primer combination for the enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection adopts FAM fluorescent groups for marking the 5' -end of a specific probe sequence of the enterocolitis yersinia universal gene foxA and the virulence gene ystB; the 5' -end of a specific probe sequence of virulence genes ail and yadA is marked by a VIC fluorescent group; the 5' -end of the specific probe sequences of virulence genes ystA and virF7 is marked by ROX fluorescent groups.
Secondly, the invention provides a yersinia enterocolitica nucleic acid multiplex fluorescence PCR detection kit, which comprises six gene detection reaction mixed liquids A and B of yersinia enterocolitica, a hot start DNA polymerase, a positive quality control product and a negative quality control product of the yersinia enterocolitica;
the reaction mixture A contains a primer and a probe for detecting the yersinia enterocolitica virulence gene foxA, ail, ystA;
the reaction mixture B contains a primer and a probe for detecting the yersinia enterocolitica virulence gene ystB, yadA, virF;
the positive quality control product is a recombinant plasmid containing detection target sequences of six genes foxA, ail, ystA, ystB, yadA, virF of yersinia enterocolitica;
the negative quality control product is purified water which does not contain RNase and DNase.
The enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection kit comprises a reaction mixed solution A and a reaction mixed solution B, and a reaction buffer solution and Mg 2+ And deoxynucleotide triphosphates; the reaction buffer is 2X One Step RT-qPCR Probe Buffer.
The storage condition of the kit is-20 ℃ and the repeated freezing and thawing times are not more than 5 times.
The kit for detecting the enterocolitis yersinia nucleic acid by multiplex fluorescence PCR has the detection lower limit of 1X 10 3 CFU/ml。
Again, the present invention provides a method of multiplex fluorescence PCR detection of yersinia enterocolitica nucleic acid using the kit of any one of claims 4-7; the PCR reaction system used for detection by the kit is 20 μl, and comprises 18 μl of reaction mixture A or reaction mixture B and 2 μl of nucleic acid sample to be detected.
In the method for detecting the enterocolitis yersinia nucleic acid by multiplex fluorescence PCR, the amount of each primer and probe in the reaction mixture A and the reaction mixture B is 0.08 mu l, 0.08 mu l and 0.04 mu l respectively.
The reaction conditions of the detection of the enterocolitis yersinia nucleic acid multiplex fluorescence PCR method are as follows: a pre-denaturation stage: 94 ℃ for 2min; amplification stage: denaturation at 94 ℃,10sec; annealing at 56 ℃ for 50sec; a total of 40 cycles; the fluorescent signal is detected during the annealing step.
The invention has the following beneficial effects:
1) The invention designs specific primers and fluorescent labeling probes for the universal gene oxA and the five virulence genes ail, ystA, ystB, yadA, virF of the yersinia enterocolitica, and develops a multiplex real-time fluorescent PCR detection kit for the nucleic acid of the yersinia enterocolitica, so as to judge whether the sample to be detected contains the yersinia enterocolitica and judge whether the sample has pathogenicity at the same time, and achieve the purpose of qualitatively detecting the yersinia enterocolitica and completing identification of virulence genes of the yersinia enterocolitica.
2) The specific primer probe and the detection method have strong specificity and high sensitivity, are simple to operate, low in cost, good in stability, time-saving, efficient and convenient compared with a single fluorescent PCR method, are suitable for laboratory emergency diagnosis and rapid typing identification of Yersinia enterocolitica, and have important significance for detection of Yersinia enterocolitica containing different virulence genes.
Drawings
FIG. 1 is a graph showing the amplification of a control product for real-time fluorescence PCR detection of the cationic nature of Yersinia enterocolitica fraction 1 (foxA gene, ail gene, ystA gene);
FIG. 2 is a graph showing the amplification of a real-time fluorescence PCR-based detection of a cationic control of Yersinia enterocolitica fraction 1 (ystB gene, yadA gene, virF gene) according to the present invention;
FIG. 3 is a graph showing the amplification of Yersinia enterocolitica foxA gene detected by real-time fluorescence PCR according to the present invention;
FIG. 4 is a graph showing the amplification of the real-time fluorescence PCR detection of yersinia enterocolitica ail gene according to the present invention;
FIG. 5 is a graph showing the amplification of the yersinia enterocolitica ystA gene by real-time fluorescence PCR detection in accordance with the present invention;
FIG. 6 is a graph showing the amplification of the real-time fluorescence PCR detection of the yersinia enterocolitica ystB gene according to the present invention;
FIG. 7 is a graph showing the amplification of the yarA gene of Yersinia enterocolitica by real-time fluorescence PCR;
FIG. 8 is a graph showing the amplification of the real-time fluorescence PCR detection of the yersinia enterocolitica virF gene;
FIG. 9 is a graph showing the amplification of nucleic acids from 7 diarrhea bacteria detected by a Yersinia enterocolitica multiplex real-time fluorescent PCR detection system, with no S-type amplification for 7 pathogens.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and the accompanying drawings, and it is obvious that the described embodiments are only preferred embodiments of the present invention, not all embodiments, nor other forms of limitation of the present invention, and any person skilled in the art may make changes or modifications and equivalent variations using the disclosed technical matters. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
Example 1
In this example, a primer combination, a kit and a method for multiplex fluorescence PCR detection of Yersinia enterocolitica nucleic acid are proposed for determining whether Yersinia enterocolitica is contained in a sample and determining whether Yersinia enterocolitica is pathogenic by detecting 6 genes, foxA, ail, ystA, ystB, yadA, virF, of Yersinia enterocolitica and determining whether Yersinia enterocolitica is contained in the sample and whether Yersinia enterocolitica is pathogenic by using the 6 genes. It should be noted that the examples set forth herein are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. The reagents used therein, such as kits, buffers, etc., are only those specifically selected in this particular example, and it is understood that those skilled in the art can select corresponding reagents of other companies as needed to achieve the object of the present invention. The method comprises the following steps:
first, primers and probes were designed and synthesized based on the 6 gene sequences of yersinia enterocolitica foxA, ail, ystA, ystB, yadA, virF, see table 1.
TABLE 1 primer and probe sequences
As shown in Table 1, in the probes designed in this example, the 5' -end of the specific probe sequences of the general gene foxA and the virulence gene ystB of Yersinia enterocolitica were labeled with FAM fluorescent groups; the 5' -end of a specific probe sequence of virulence genes ail and yadA is marked by a VIC fluorescent group; the 5' -end of the specific probe sequences of virulence genes ystA and virF7 is marked by ROX fluorescent groups.
The kit for detecting the enterocolitis yersinia nucleic acid multiplex fluorescence PCR provided by the embodiment is a multiplex real-time fluorescence PCR detection kit capable of detecting the enterocolitis yersinia universal gene foxA and five virulence genes ail, ystA, ystB, yadA, virF genes simultaneously. The kit comprises six kinds of enterocolitis yersiniaGene detection reaction mixture A and B, hot start DNA polymerase and yersinia enterocolitica positive quality control and negative quality control. The reaction mixture contains reaction buffer solution and Mg 2+ And deoxynucleotide triphosphate mixture (dNTP), specific primers of six genes of yersinia enterocolitica, taqMan fluorescent probes and the like; the different primer probes in the two groups of reaction mixed solutions are respectively: the reaction mixture A contains foxA, ail, ystA; the reaction mixture B contains ystB, yadA, virF; the positive quality control is a recombinant plasmid (without biological hazard) containing the detection target sequences of the six genes of the yersinia enterocolitica; the negative quality control material is RNase-free and DNase water.
The PCR reaction system adopted by the kit is 20 μl,18 μl of the reaction mixture A or the reaction mixture B, and 2 μl of the nucleic acid sample to be detected. The reaction mixture A and the reaction mixture B specifically comprise: 2 XOne Step RT-qPCR Probe Buffer. Mu.l, 0.08. Mu.l and 0.04. Mu.l of each primer and probe, respectively, 0.4. Mu.l of hot-start DNA polymerase, and the balance of sterilized water.
PCR reaction procedure for kit: a pre-denaturation stage: 94 ℃ for 2min; amplification stage: denaturation at 94 ℃,10sec; annealing at 56 ℃ for 50sec; for a total of 40 cycles. The fluorescent signal is detected during the annealing step.
And (3) quality control: negative and positive controls are set up in each experiment, the negative control has no Ct value (or Ct value is 0), the Ct value of the positive control is less than or equal to 30, otherwise, the experimental result is not established.
Interpretation of the results:
positive: an S-shaped amplification curve appears, and the Ct value is less than or equal to 35; suspicious: an "S" type amplification curve occurs, but Ct values > 35; negative: the "S" type amplification curve does not appear, or the curve is not "S" type although exceeding the threshold; for suspicious results, the experiment should be repeated, and if the repeated experiment or the S-shaped amplification curve appears, the negative control has no pollution, and can be judged to be positive.
Sample type and requirements: the method mainly comprises fecal samples, isolated strains or enrichment cultures and the like, and adopts commercial kits with stable and reliable performance, and the specific method is referred to the instruction book of the corresponding commercial kit; the extracted nucleic acid should be detected immediately, otherwise, the nucleic acid should be kept at-80 ℃ to-20 ℃ after being packaged.
The kit has storage condition of-20deg.C and repeated freezing and thawing times of no more than 5 times, and has detection limit of 1×10 for Yersinia enterocolitica nucleic acid 3 CFU/ml。
Example 2
This example is the construction and validation process of the multiplex real-time fluorescent PCR detection kit described in example 1. The method comprises the following steps:
1. design and synthesis of primers and TaqMan probes
The 6 gene sequences of yersinia enterocolitica foxA, ail, ystA, ystB, yadA, virF in Genbank and domestic and foreign literature were analyzed using NCBI Blast tool, and stable conserved regions thereof were selected as detection target sequences, respectively, and primers and probes were designed and synthesized for the detection target sequences (see Table 1). Both primers and probes were synthesized by the company Shanghai, inc.
Wherein the 5' -end of the specific probe sequences of foxA and ystB is marked by FAM fluorescent groups; the 5' -end of the specific probe sequence of the ail and yadA target 2 is marked by a VIC fluorescent group; the 5' -end of the specific probe sequences of ystA and virF are marked by ROX fluorescent groups. The 5 'end fluorescent group of the specific probe can be selected from FAM/VIC (or HEX) ROX/CY5 and other different fluorescent labels, and the 3' end can be correspondingly selected from corresponding quenching groups.
2. Preparation for detecting bacterial species
The enterocolitis yersinia (ATCC 23715, CMCC52225, 52217) used in this example was purchased from chinese industrial microorganism strain collection management center as well as other negative controls including escherichia coli O157 (ATCC 35150), salmonella paratyphi (CMCC (B) 50094), vibrio parahaemolyticus (ATCC 17802), shigella flexneri (ATCC 12022), vibrio vulnificus (ATCC 27562), bacillus cereus (ATCC 11778), campylobacter coli (CICC 24753).
3. Extraction of nucleic acid from strains
The nucleic acid of the strain is extracted by using a nucleic acid extraction or purification reagent produced by Jiangsu and Chuangxiong biotechnology Co. Specific steps refer to the kit operating instructions.
4. Screening of primers and probes
The primers and probes are adopted to detect the extracted yersinia enterocolitica nucleic acid, and the primer probe combination with optimal sensitivity, specificity and repeatability is screened out through repeated experiments. (see sequence Listing 1)
5. Optimization of reaction conditions
The elements of the primer, the probe, the enzyme and the like are optimized one by one, and a determined reaction system is as follows: 2 XOne Step RT-qPCR Probe Buffer IV. Mu.l, 100. Mu. Mol/L primer each 0.5. Mu.l, 100. Mu. Mol/L probe 0.2. Mu.l, enzyme cocktail 2. Mu.l, template 2. Mu.l, and sterile water were added to a final system of 20. Mu.l.
6. Amplification procedure
According to the length of the amplified fragment, the annealing temperature of the primer and the probe and the enzyme characteristics, the annealing temperature and the reaction time of the reaction are mainly optimized, and a rapid amplification program is determined. The final determined cycle parameters were: a pre-denaturation stage: 94 ℃ for 2min; amplification stage: denaturation at 94 ℃,10sec; annealing at 56 ℃ for 50sec; for a total of 40 cycles. The fluorescent signal is detected during the annealing step. After amplification, the data were analyzed under the same conditions to determine Ct values for each sample.
7. Evaluation of detection Limit
The detection limit of the kit provided by the invention is evaluated by the positive bacteria standard, and the concentration of the positive standard is as follows: 1X 106CFU/ml to 1X 107CFU/ml, the lower limit of detection of yersinia enterocolitica nucleic acid by the kit described in example 1 was verified to be 1X 10 3 CFU/ml。
8. Evaluation of detection specificity
As shown in fig. 1 to 9, in this example, the specificity of the kit described in example 1 was evaluated using the above-described yersinia enterocolitica nucleic acid as a template, and clear amplification curves were seen for each detection of yersinia enterocolitica; and no positive amplification curve was generated using the 7 other common diarrhea bacteria assays described above, indicating that there was no cross-reaction between the probes and primers described in example 1 and the other strains selected.
Although certain embodiments of the present invention have been described above by way of example in a preferred manner, it will be understood by those skilled in the art that the present invention is not limited to the embodiments disclosed above, but may be modified in light of the knowledge of those skilled in the art to which the present invention pertains without exceeding the scope of the claimed invention. For example, the fluorescent real-time PCR used in the present invention may also employ, as required, a labeling substance other than the fluorescent reporter group and the fluorescent quenching group indicated in examples listed in the specification, such as a labeling substance of FAM, HEX, JOE, ROX, cy or the like; or other labeling systems than Taqman technology, such as fluorescent probe labeling technologies, e.g., molecular beacon MB probes, scorpion probes, fluorescent double hybridization probes, etc.; or using dye-embedding method such as SYBR Green I and LC Green and other saturated dyes, and qualitatively or quantitatively detecting the existence of the target gene only by using the specific primer sequence and the primer ratio, thereby rapidly and specifically detecting the existence of the yersinia enterocolitica foxA, ail, ystA, ystB, yadA, virF gene in the same reaction system. Therefore, such modifications and alternatives as would be apparent to one skilled in the art are intended to fall within the scope of the present invention. The scope of the invention should be defined by the appended claims.
Claims (10)
1. A primer combination for yersinia enterocolitica nucleic acid multiplex fluorescence PCR detection, characterized in that: comprises six primer pairs and six probes for detecting yersinia enterocolitica general gene foxA and five virulence genes ail, ystA, ystB, yadA, virF genes; the nucleotide sequence of each primer and each probe is shown as SEQ NO. 1-SEQ NO. 18.
2. The primer combination for the multiplex fluorescence PCR detection of yersinia enterocolitica nucleic acid according to claim 1, wherein: wherein,
the primer pair and the probe shown in SEQ NO. 1-SEQ NO.3 are used for detecting the gene foxA;
the primer pair and the probe shown in SEQ NO. 4-SEQ NO.6 are used for detecting virulence genes ail;
the primer pair and the probe shown in SEQ NO. 7-SEQ NO.9 are used for detecting virulence genes ystA;
the primer pair and the probe shown in SEQ NO. 10-SEQ NO.12 are used for detecting virulence genes ystB;
the primer pair and the probe shown in SEQ NO. 13-SEQ NO.15 are used for detecting virulence genes yadA;
the primer pair and the probe shown in SEQ NO. 16-SEQ NO.18 are used for detecting virulence gene virF.
3. The primer combination for the multiplex fluorescence PCR detection of yersinia enterocolitica nucleic acid according to claim 1, wherein: the 5' -end of a specific probe sequence of the general gene foxA and the virulence gene ystB of yersinia enterocolitica is marked by FAM fluorescent groups; the 5' -end of a specific probe sequence of virulence genes ail and yadA is marked by a VIC fluorescent group; the 5' -end of the specific probe sequences of virulence genes ystA and virF7 is marked by ROX fluorescent groups.
4. A yersinia enterocolitica nucleic acid multiplex fluorescence PCR detection kit, characterized in that: comprises six kinds of gene detection reaction mixed solutions A and B of yersinia enterocolitica, a hot start DNA polymerase, a positive quality control product and a negative quality control product of the yersinia enterocolitica;
the reaction mixture A contains the gene for detecting the yersinia enterocolitica virulencefoxA、ail、ystAIs a primer and a probe of (a);
the reaction mixture B contains the gene for detecting the yersinia enterocolitica virulenceystB、yadA、virFIs a primer and a probe of (a);
the positive quality control product is a recombinant plasmid containing detection target sequences of six genes foxA, ail, ystA, ystB, yadA, virF of yersinia enterocolitica;
the negative quality control product is purified water which does not contain RNase and DNase.
5. The small intestine according to claim 4The yersinia colitis nucleic acid multiplex fluorescence PCR detection kit is characterized in that: the reaction mixed solution A and B also contains reaction buffer solution and Mg 2+ And deoxynucleotide triphosphates;
the reaction buffer is 2X One Step RT-qPCR Probe Buffer.
6. The yersinia enterocolitica nucleic acid multiplex fluorescence PCR detection kit of claim 4, wherein: the storage condition of the kit is-20 ℃, and the repeated freezing and thawing times are not more than 5 times.
7. The yersinia enterocolitica nucleic acid multiplex fluorescence PCR detection kit of claim 4, wherein: the lower limit of detection of the kit on the enterocolitis yersinia nucleic acid is 1 multiplied by 10 3 CFU/ml。
8. A method for detecting yersinia enterocolitica nucleic acid multiplex fluorescence PCR, which is characterized in that: performing a test using the kit of any one of claims 4-7; the PCR reaction system for detection and selection by using the kit is 20 mu l and comprises 18 mu l of reaction mixed liquid A or reaction mixed liquid B, and nucleic acid samples to be detected are 2 mu l.
9. The method for the multiplex fluorescence PCR detection of yersinia enterocolitica nucleic acid according to claim 4, wherein: and each primer and each probe in the reaction mixed solution A and the reaction mixed solution B are respectively 0.08 mu l, 0.08 mu l and 0.04 mu l.
10. The method for the multiplex fluorescence PCR detection of yersinia enterocolitica nucleic acid according to claim 4, wherein: the reaction conditions for this detection were: a pre-denaturation stage: 94 ℃ for 2min; amplification stage: denaturation at 94 ℃,10sec; annealing at 56 ℃ for 50sec; a total of 40 cycles; the fluorescent signal is detected during the annealing step.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311809361.6A CN117757963A (en) | 2023-12-26 | 2023-12-26 | Primer combination, kit and method for enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311809361.6A CN117757963A (en) | 2023-12-26 | 2023-12-26 | Primer combination, kit and method for enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117757963A true CN117757963A (en) | 2024-03-26 |
Family
ID=90310380
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311809361.6A Pending CN117757963A (en) | 2023-12-26 | 2023-12-26 | Primer combination, kit and method for enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117757963A (en) |
-
2023
- 2023-12-26 CN CN202311809361.6A patent/CN117757963A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mangal et al. | Molecular detection of foodborne pathogens: A rapid and accurate answer to food safety | |
| Chen et al. | PCR-based methodologies for detection and characterization of Listeria monocytogenes and Listeria ivanovii in foods and environmental sources | |
| Chiang et al. | Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157: H7, Vibrio parahaemolyticus, Salmonella spp. and Pseudomonas fluorescens in milk and meat samples | |
| CA2596059C (en) | Method of quantitatively analysing microorganism targeting rrna | |
| US8268984B2 (en) | Detection of salmonella by real-time multiplex PCR | |
| KR102618075B1 (en) | Composition for rapid detection of Salmonella D group in feed containing oligonucleotide set | |
| US20100075305A1 (en) | Detection of bacterium by utilizing dnaj gene and use thereof | |
| AU2013205010B2 (en) | Detection of Shiga toxin genes in bacteria | |
| US9024002B2 (en) | Compositions and methods for detection of Salmonella species | |
| US20250034660A1 (en) | Methods and Compositions for Determining Salmonella Presence and Concentration Using PCR Primers of Varying Amplification Efficiencies | |
| US20030113757A1 (en) | Rapid and specific detection of campylobacter | |
| Hossain et al. | DNA-based methods for species identification in food forensic science | |
| MXPA03012075A (en) | Methods and oligonucleotides for the detection of $i(salmonella) sp., $i(e. coli) o157:h7, and $i(listeria monocytogenes ). | |
| Lei et al. | Duplex detection of Vibrio Cholerae and Vibrio Parahaemolyticus by real-time recombinase polymerase amplification | |
| US20230227921A1 (en) | Methods and Compositions for Detecting Virulent and Avirulent Escherichia coli Strains | |
| CN111020039A (en) | Species-specific molecular target of Campylobacter jejuni and its rapid detection method | |
| KR101299627B1 (en) | Primers for simultaneous detection of foodborne bacteria and use thereof | |
| CN117757963A (en) | Primer combination, kit and method for enterocolitis yersinia nucleic acid multiplex fluorescence PCR detection | |
| CN118773348B (en) | A joint detection kit for eight foodborne pathogens based on multiple probe melting curve method | |
| Rusul et al. | Molecular Methods for the Detection and Characterization of Food‐Borne Pathogens | |
| Fratamico et al. | Applications of the polymerase chain reaction for detection, identification, and typing of food-borne microorganisms | |
| KR20200059765A (en) | Development of a Singleplex Real-Time PCR Kit for Rapid Detection of Cronobacter sakazakii ompA target gene | |
| US20060240442A1 (en) | Methods and oligonucleotides for the detection of Salmonella SP., E coli 0157:H7, and Listeria monocytogenes | |
| US20240141444A1 (en) | Methods and compositions for determining microorganism presence and concentration using pcr primers of varying amplification efficiencies | |
| Sanderson et al. | Genetic techniques: PCR, NASBA, hybridisation and microarrays |
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 |