CN115141897A - A primer probe, kit and application for detecting pathogens of neonatal infection - Google Patents

Abstract

The invention discloses a primer probe and a kit for detecting pathogen of neonatal infection and application thereof. The pathogen of neonatal infection includes mycoplasma hominis, mycoplasma urealyticum, mycoplasma genitalium, chlamydia trachomatis, ureaplasma parvum, escherichia coli, streptococcus group B, gonococcus, enterovirus, cytomegalovirus or herpes simplex virus type 1. The invention designs specific primers and double probes aiming at 11 pathogens related to neonatal infection to solve the problem that one probe has a low hybridization signal. By combining the suspension microbead liquid-phase chip technology, the genotype detection of 11 pathogens can be completed in one experiment, and the sensitivity and the specificity are good. The detection kit provided by the invention has sensitive reaction and can quickly and accurately detect 11 pathogens causing genital tract infection.

Description

A primer probe, kit and application for the detection of neonatal infectious pathogens

technical field

The invention belongs to the field of molecular biology, and in particular relates to a primer probe, a kit and application for the detection of neonatal infectious pathogens.

Background technique

A newborn is an infant who is less than 28 days old after birth. Newborns with symptoms of infection at birth account for about 10% of the birth rate of newborns.

Numerous studies have shown that neonatal infections are mainly caused by perinatal reproductive tract infections in pregnant women. After infection in the maternal reproductive tract, pathogens can infect the placenta through blood, causing damage to the chorionic and capillary endothelium, destroying the placental barrier, and entering the fetus to cause miscarriage, embryonic development, and fetal malformation. For example, bacterial vaginosis during pregnancy can cause miscarriage, premature rupture of membranes, premature birth, amniotic fluid infection, postpartum endometritis, and post cesarean incision infection. Genital herpes simplex virus infection: infection in the first trimester can cause fetal malformation; infection before 20 weeks of pregnancy, the miscarriage rate is 34%; the virus infection rate of newborns born vaginally by pregnant women infected with the virus is 50%, and the mortality rate of neonatal herpes infection is as high as More than 70%, most of them develop disease 4-7 days after birth, and die in 10-14 days due to the deterioration of general condition, and most of the survivors are left with central nervous system sequelae.

We also found a large number of related pathogens in clinical diagnostic testing work, from which we isolated and collected 11 pathogenic pathogens that commonly cause maternal reproductive tract infections during the perinatal period, Mycoplasma hominis (MH), Ureaplasma urealyticum ( UU), Mycoplasma genitalium (MG), Chlamydia trachomatis (CT), Ureaplasma parvum (UP), Escherichia coli (E.coli), Group B Streptococcus (GBS), Neisseria gonorrhoeae (NG), intestinal virus (EVS), cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-I). These pathogens have not yet been included in the perinatal screening items, and a monitoring and diagnostic system is urgently needed in clinical practice. Based on the clinical problems, this project plans to develop and construct a multi-channel rapid nucleic acid detection system for 11 pathogens infected by neonates based on the integrated multiplex PCR amplification and liquid-phase hybridization integrated nucleic acid detection technology for pathogens. This method avoids a large number of repetitions of a single PCR assay and enables specific detection of 11 target pathogens, while retaining the advantages of PCR's high sensitivity.

PCR method is the gold standard for pathogen detection, which has greatly improved the sensitivity and specificity of clinical pathogen detection. The current fluorescence quantitative PCR platform can realize automatic and high-sensitivity screening. However, the disadvantage of the PCR method is that the detection throughput of one tube is limited, and more than 10 pathogens cannot be detected in one tube at a time. Faced with many problems, there are some advanced technologies and products on the market. For example, the development of PCR chip technology can realize multiple detection, such as Mérieux's Filmarray platform and Saipei GeneXpert platform. However, these platforms require imported special equipment. The biggest drawback is that the cost of reagents is expensive, the throughput is limited, and the universality is not strong. At present, there are fluorescent quantitative PCR detection kits on the market to detect GBS, Ureaplasma urealyticum (UU), CT and MG. With the development of technology, my country has developed triple, quadruple and ten-fold detection systems for reproductive tract infections, mainly focusing on sexually transmitted disease (Sexually Transmitted Disease, STD) pathogens. However, the multiplex detection system for pathogens for neonatal infection has not yet been reported, and cannot achieve wide coverage, universal screening, and early prevention and treatment. There is an urgent need to develop a multiplex detection system that can identify pathogens at high risk of neonatal infection.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the defects in the prior art, and to provide a primer, a probe, a kit and application for the detection of neonatal infectious pathogens.

In order to qualitatively detect 11 kinds of reproductive tract pathogens in one tube, the present invention provides 1. Using a tube of multiplex PCR Mix to cover the amplification of 11 kinds of pathogens, and a suspension microbead liquid-phase chip kit that combines primers and probes 2. The multiplex PCR Mix contains 11 pairs of complete amplification primers, and the amplification product of each pair of primers corresponds to a specific site of a pathogen; 3. The system designs a double probe for the amplification product of each pathogen Needle, that is, both amplification products have a complementary probe (sense strand probe S and antisense strand probe A), and both probes are coupled to a magnetic bead, which can amplify the product. signal enhancement.

Therefore, the first object of the present invention is to provide a multiplex PCR detection primer and probe set for detecting 11 neonatal infectious pathogens, which include:

Primers for detecting the nucleotide sequences of Mycoplasma hominis shown in SEQ ID NO. 1-2 and probes for the nucleotide sequences shown in SEQ ID NO. 3-4;

Primers for detecting the nucleotide sequences of Ureaplasma urealyticum shown in SEQ ID NO. 5-6 and probes for the nucleotide sequences shown in SEQ ID NO. 7-8;

Primers for the nucleotide sequences shown in SEQ ID NO. 9-10 and probes for the nucleotide sequences shown in SEQ ID NO. 11-12 for detecting Mycoplasma genitalium;

Primers for the nucleotide sequences shown in SEQ ID NO. 13-14 and probes for the nucleotide sequences shown in SEQ ID NO. 15-16 for detecting Chlamydia trachomatis;

Primers for the nucleotide sequences shown in SEQ ID NO. 17-18 and probes for the nucleotide sequences shown in SEQ ID NO. 19-20 for detecting Ureaplasma parvum;

Primers for detecting the nucleotide sequences shown in SEQ ID NO. 21-22 and probes for the nucleotide sequences shown in SEQ ID NO. 23-24 for detecting Escherichia coli;

Primers for detecting the nucleotide sequences shown in SEQ ID NO. 25-26 and probes for the nucleotide sequences shown in SEQ ID NO. 27-28 for detection of group B Streptococcus;

Primers for detecting nucleotide sequences shown in SEQ ID NO. 29-30 and probes for nucleotide sequences shown in SEQ ID NO. 31-32 for detection of gonococcus;

Primers for the nucleotide sequences shown in SEQ ID NO.33-34 and probes for the nucleotide sequences shown in SEQ ID NO.35-36 for detecting enteroviruses;

Primers for the nucleotide sequences shown in SEQ ID NOs. 37 to 38 and probes for the nucleotide sequences shown in SEQ ID NOs. 39 to 40 for detection of cytomegalovirus; and

The primers for detecting the nucleotide sequences shown in SEQ ID NO. 41 to 42 and the probes for the nucleotide sequences shown in SEQ ID NOs. 43 to 44 are used for the detection of herpes simplex virus type 1.

Preferably, the 5' ends of the upstream and downstream primers of the detection primer are all modified with Biotin.

Preferably, the 5' ends of the detection probes are all modified with 5'-Amino C6+Spacer 18 amino groups.

The second object of the present invention is to provide the application of the detection primers and probe sets in the preparation of detection products for neonatal infection pathogens.

Preferably, the neonatal infection pathogens include Mycoplasma hominis, Mycoplasma urealyticum, Mycoplasma genitalium, Chlamydia trachomatis, Ureaplasma parvum, Escherichia coli, Group B streptococcus, Neisseria gonorrhoeae, enterovirus, cytomegalo virus or herpes simplex virus type 1.

The third object of the present invention is to provide a gene chip for detecting 11 kinds of neonatal infectious pathogens, wherein the fixed carrier of the chip contains SEQ ID NO. SEQ ID NO.11～12, SEQ ID NO.15～16, SEQ ID NO.19～20, SEQ ID NO.23～24, SEQ ID NO.27～28, SEQ ID NO.31～32, SEQ ID NO Probes shown in .35-36, SEQ ID NO.39-40 and SEQ ID NO.43-34.

Preferably, the immobilization carrier is a magnetic microbead whose surface is modified with carboxyl groups.

The fourth object of the present invention is to provide the application of the gene chip in the preparation of a detection product for neonatal infection pathogens.

The fifth object of the present invention is to provide a kit for detecting 11 neonatal infectious pathogens, the kit comprising the detection primers and probe sets.

Preferably, the kit further includes detection reagents and nucleic acid extraction reagents.

This kit targets 11 pathogens related to neonatal infection: Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), Mycoplasma genitalium (MG), Chlamydia trachomatis (CT), Ureaplasma parvum (UP), Escherichia coli Design specific primers for E.coli, group B streptococcus (GBS), gonococcus (NG), enterovirus (EVS), cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-Ⅰ) With double probes, the samples in the same tube are amplified by multiplex PCR, and the labeled PCR products are obtained by PCR. A pathogen-specific dual probe is covalently cross-linked to the same microsphere. The labeled PCR product is hybridized with the microspheres, and a chromogenic reagent is added for detection on the machine. The detection result is judged according to the level of the signal of the corresponding probe of the pathogen.

The beneficial effects of the present invention are: the present invention designs specific primers and double probes for 11 pathogens related to neonatal infection, so as to solve the problem of low hybridization signal of one probe. Combined with the suspension microbead liquid-phase chip technology, the genotype detection of 11 pathogens can be completed in one experiment, with good sensitivity and specificity. The detection kit provided by the invention has a sensitive response and can quickly and accurately detect 11 kinds of pathogens that cause reproductive tract infection.

Description of drawings

Figure 1 shows the effect of single and double probes on the hybridization signal.

Detailed ways

The present invention will be further described in detail below through the examples, which are only used to illustrate the present invention, but do not limit the scope of the present invention.

Example 1

1. Design of multiplex PCR primers and probes:

The present invention targets 11 pathogens related to neonatal infection: Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), Mycoplasma genitalium (MG), Chlamydia trachomatis (CT), Ureaplasma parvum (UP), Escherichia coli Design specific primers and With double probes, the samples in the same tube are amplified by multiplex PCR, and the labeled PCR products are obtained by PCR. A pathogen-specific dual probe is covalently cross-linked to the same microsphere. The labeled PCR product is hybridized with the microspheres, and a chromogenic reagent is added for detection on the machine. The detection result is judged according to the level of the signal of the pathogen-corresponding probe-coupled microspheres.

The primer sequences involved in the present invention are shown in Table 1 below:

Table 1. Primer sequences for multiplex PCR detection

The 5' ends of the upstream and downstream primers are all modified with Biotin.

The corresponding double probe sequences corresponding to the 11 pathogen gene loci are shown in Table 2 below:

Table 2. Dual-probe sequences for detection of 11 pathogenic gene loci by suspension microbead liquid chip

2. Preparation of suspension microbead liquid phase chip:

The magnetic microbeads modified with carboxyl groups on the surface were purchased from Luminex Corporation of the United States, and the probes were coupled to the magnetic beads according to the probe magnetic bead coupling scheme provided by the Luminex Corporation of the United States. The dual probe of each pathogen is coupled with one encoded magnetic bead, and a total of 22 probes for 11 pathogens to be detected are coupled with a total of 11 encoded magnetic beads. The specific process is as follows: take out 40 μL (1×10 ⁷ ) magnetic microspheres, adsorb the magnetic microspheres with a magnetic rod, aspirate the supernatant, add 5 μL of 0.1M MES (pH 4.5), and mix well. Each of the two probes used was diluted to 100 μM and 1 μL of each was added to the coupling system. For the first time, 2.5 μL of 10 mg/mL EDC was added, and after mixing, it was placed in the dark for 30 min, and vortexed twice during this period. Repeatedly add EDC (2.5 μL, 10 mg/mL) again, mix well and place in the dark for 30 min, during which time vortex twice. Wash once with 500 μL of 0.02% Tween and 0.1% SDS each, and finally resuspend the microspheres in 80 μL of TE (pH 8.0) solution. After mixing, use a hemocytometer to count the number of microspheres (after counting the number of 4 large squares in the four corners) Converted to the number of microspheres per microliter), the concentration of each microsphere should reach more than 150/μL. Store at 4°C in the dark. Mix the microspheres of the 11 coupled probes in equal volume ratios, and then use 1.5×TMAC, 1×TE, and the mixed microspheres to form a hybridization Mix at a volume ratio of 33:10:2, totaling 45 μL/person share.

3. PCR test the sample:

Using the single-tube multiplex PCR amplification system in the present invention, the detection of Mycoplasma hominis (MH), Mycoplasma urealyticum (UU), Mycoplasma genitalium (MG), Chlamydia trachomatis (CT), Ureaplasma parvum (UP), Escherichia coli Helicobacter (E.coli), Group B Streptococcus (GBS), Neisseria gonorrhoeae (NG), Enterovirus (EVS), Cytomegalovirus (CMV), Herpes Simplex Virus 1, Type 1 (HSV-Ⅰ) 10 each A sequenced positive sample was tested. The PCR reaction system was a total volume of 20 μL, including 2 μL of DNA template with a concentration of 10 ng/μL, 2 μL of 10× PCR reaction buffer (containing Mg ²⁺ ), 3 μL of 5M Betain, and 1 μL of enzyme system (including 0.5 μL of 40 mM dNTP mixed solution). μL, 5U/μL hot-start Taq enzyme 0.3 μL, 1U/μL UDG enzyme 0.1 μL, 10mM dUTP 0.1 μL); the final concentration of upstream and downstream primers is 0.25 μM, and deionized water is supplemented to 20 μL.

The reaction was carried out on the T-100 PCR amplicon of BioRad Company. The reaction conditions were 50°C, 3 minutes, 95°C, 10 minutes, and 45 cycles of 94°C, 30 seconds, 56°C, 60 seconds, 72°C, 30 seconds. seconds; 72 ℃, 5 minutes, 12 ℃ storage.

4. On-board testing and result analysis:

Take 2.5 μL of PCR product and 45 μL of coupled microbeads, the total reaction volume is 47.5 μL, after sealing, carry out the hybridization reaction according to the following conditions: denaturation at 95°C for 5 minutes, and then hybridize at 60°C for 15 minutes. Aspirate the color developing working solution again, add it to the hybridization reaction solution, 25 μL per reaction, mix by pipetting, and incubate the color developing reaction at 60°C for 7 min in the dark. The hybridization products were detected on the machine, and the detection instrument was a Luminex liquid-phase chip detector MAGPIX. The reference interval of the signal value of each pathogen detection probe is shown in Table 3 below:

Table 3. Signal value reference interval of each pathogen detection probe

When the sum of the detection signal values of the double probes coupled on a microsphere is greater than or equal to the positive signal value, the site is judged to be positive.

When the sum of the detection signal values of the double probes coupled on a microsphere is less than the negative signal value, the site is judged to be negative.

When the sum of the detection signal values of the double probes coupled on a microsphere is between the negative and positive signal values, the site cannot be determined and needs to be further confirmed (repeat the experiment or use other methods to detect).

The results show that the single-tube multiplex PCR amplification system of the present invention is used to detect the sequenced positive samples of the above 11 pathogens, and the sensitivity and specificity of each pathogen are high, as shown in Table 4.

Table 4. Accuracy data of the present invention for detecting 11 pathogens

Example 2

Since in the hybridization system of the present invention, two amplification products are designed with double probes, the signal of the target pathogen can be better collected. The single-probe system can only hybridize one target product in the PCR product. For pathogens with relatively low load, the detection result may fall in the gray area, which is not easy to judge. The dual probe system can hybridize two products in the same PCR amplification product to enhance the detection signal, thereby solving the problem of low signal. The single probe and double probe were the same in all reaction conditions, see Example 1 for details. Table 5 shows the single probe and double probe information of 11 pathogens. The influence of single-probe and dual-probe hybridization signal values for each pathogen is shown in Table 6 and Figure 1. Taking 11 pathogens as examples, the effects of single probe and double probe on hybridization signal are shown in Figure 1.

Table 5. Single-probe and dual-probe information for the present invention to detect 11 pathogens

Table 6. The single-probe and double-probe hybridization signal values of the present invention for detecting 11 pathogens

The above-mentioned embodiments are only to describe the preferred embodiments of the present invention, not to limit the scope of the present invention. Such deformations and improvements shall fall within the protection scope determined by the claims of the present invention.

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Claims (10)

1. a multiplex PCR detection primer, probe group for detecting 11 kinds of neonatal infection pathogens, is characterized in that, comprises: Primers for detecting the nucleotide sequences of Mycoplasma hominis shown in SEQ ID NO. 1-2 and probes for the nucleotide sequences shown in SEQ ID NO. 3-4; Primers for detecting the nucleotide sequences of Ureaplasma urealyticum shown in SEQ ID NO. 5-6 and probes for the nucleotide sequences shown in SEQ ID NO. 7-8; Primers for the nucleotide sequences shown in SEQ ID NO. 9-10 and probes for the nucleotide sequences shown in SEQ ID NO. 11-12 for detecting Mycoplasma genitalium; Primers for the nucleotide sequences shown in SEQ ID NO. 13-14 and probes for the nucleotide sequences shown in SEQ ID NO. 15-16 for detecting Chlamydia trachomatis; Primers for the nucleotide sequences shown in SEQ ID NO. 17-18 and probes for the nucleotide sequences shown in SEQ ID NO. 19-20 for detecting Ureaplasma parvum; Primers for detecting the nucleotide sequences shown in SEQ ID NO. 21-22 and probes for the nucleotide sequences shown in SEQ ID NO. 23-24 for detecting Escherichia coli; Primers for detecting the nucleotide sequences shown in SEQ ID NO. 25-26 and probes for the nucleotide sequences shown in SEQ ID NO. 27-28 for detection of group B Streptococcus; Primers for detecting nucleotide sequences shown in SEQ ID NO. 29-30 and probes for nucleotide sequences shown in SEQ ID NO. 31-32 for detection of gonococcus; Primers for the nucleotide sequences shown in SEQ ID NO.33-34 and probes for the nucleotide sequences shown in SEQ ID NO.35-36 for detecting enteroviruses; Primers for the nucleotide sequences shown in SEQ ID NOs. 37 to 38 and probes for the nucleotide sequences shown in SEQ ID NOs. 39 to 40 for detection of cytomegalovirus; and The primers for detecting the nucleotide sequences shown in SEQ ID NO. 41 to 42 and the probes for the nucleotide sequences shown in SEQ ID NOs. 43 to 44 are used for the detection of herpes simplex virus type 1. 2. the multiplex PCR detection primers, probe sets for detecting 11 kinds of neonatal infectious pathogens according to claim 1 are characterized in that, the 5 ' ends of the upstream and downstream primers of the detection primers all have Biotin modification . 3. the multiplex PCR detection primers, the probe set for detecting 11 kinds of neonatal infection pathogens according to claim 1, it is characterized in that, the 5 ' end of described detection probe all has 5 '-Amino C6 +Spacer 18 amino modification. 4. The application of the detection primers and probe sets according to claim 1 in the preparation of detection products for neonatal infection pathogens. 5. application according to claim 5, is characterized in that, described neonatal infection pathogen comprises Mycoplasma hominis, Ureaplasma urealyticum, Mycoplasma genitalium, Chlamydia trachomatis, Ureaplasma parvum, Escherichia coli, B family Streptococcus, gonococcus, enterovirus, cytomegalovirus, or herpes simplex virus type 1. 6. A gene chip for detecting 11 kinds of neonatal infectious pathogens, wherein the fixed carrier of the chip contains SEQ ID NO.3-4, SEQ ID NO.7-8, SEQ ID NO. .11～12, SEQ ID NO.15～16, SEQ ID NO.19～20, SEQ ID NO.23～24, SEQ ID NO.27～28, SEQ ID NO.31～32, SEQ ID NO.35～ 36. Probes shown in SEQ ID NO. 39-40 and SEQ ID NO. 43-34. 7 . The gene chip according to claim 6 , wherein the immobilized carrier is a magnetic microbead whose surface is modified with carboxyl groups. 8 . 8. The application of the gene chip according to claim 6 in the preparation of a product for detecting a pathogen of neonatal infection. 9 . A kit for detecting 11 neonatal infectious pathogens, wherein the kit comprises the detection primers and probe sets of claim 1 . 10 . 10. The kit according to claim 9, further comprising detection reagents and nucleic acid extraction reagents.

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