Multiplex fluorescent quantitative PCR (polymerase chain reaction) primer probe combination for detecting common pathogenic bacteria in vaginal secretion and application of multiplex fluorescent quantitative PCR primer probe combination

Abstract

The invention relates to a multiplex fluorescent quantitative PCR primer probe combination for detecting common pathogenic bacteria in vaginal secretion and application of the multiplex fluorescent quantitative PCR primer probe combination, and belongs to the technical field of biology. The primer probe combination provided by the invention contains any one of primer probe groups 1-7. A specific primer probe combination is designed for a cfb gene and a dits gene of streptococcus agalactiae, a sapB2 gene and a cadF gene of campylobacter fetus, a nuc gene of staphylococcus aureus, an hlyA gene of listeria monocytogenes and a ureaplasma ure gene, and a multiplex fluorescent quantitative PCR detection kit and method are established and used for screening common neonatal pathogenic bacteria carried in the birth canal of a puerpera. Seven pathogenic bacteria can be detected at the same time, the specificity is 100%, the sensitivity reaches 85% or above, the minimum detection limit reaches the femtogram level, clinical intervention and early intervention can be fed back in time, and the situation of neonatal infection is avoided.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a multiplex fluorescent quantitative PCR primer-probe combination for detecting common pathogens in vaginal secretions and an application thereof. Background Art

[0002] Existing technologies for prenatal pathogen screening primarily include PCR and culture methods for Streptococcus agalactiae, and fluorescent quantitative PCR for Ureaplasma. However, newborn infections with Staphylococcus aureus are frequently reported, and reports of newborn infections with Listeria monocytogenes and Campylobacter fetus are also common abroad. Therefore, existing conventional technologies are no longer sufficient for clinical prenatal pathogen screening in pregnant women.

[0003] Since Streptococcus agalactiae, Ureaplasma, Staphylococcus aureus, Listeria monocytogenes and Campylobacter fetus are all common pathogens of neonatal infection, most of the single-plex PCR methods for these pathogens have low specificity and sensitivity. Therefore, it is necessary to establish a multiplex fluorescence quantitative PCR kit for simultaneous detection to further improve the specificity and sensitivity of the detection for routine prenatal screening of these pathogens. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a multiplex fluorescent quantitative PCR primer probe combination and application thereof for detecting common pathogens in vaginal secretions.

[0005] To achieve the above object, the technical solution adopted by the present invention is: In a first aspect, the present invention provides a primer-probe combination, wherein the primer-probe combination comprises any one of primer-probe groups 1 to 7; The nucleotide sequences of the primers in the primer-probe set 1 are shown in SEQ ID NOs: 8 and 9, and the nucleotide sequence of the probe is shown in SEQ ID NO: 10; The nucleotide sequences of the primers in the primer probe set 2 are shown in SEQ ID NOs: 11 and 12, and the nucleotide sequence of the probe is shown in SEQ ID NO: 13; The nucleotide sequences of the primers in the primer probe set 3 are shown in SEQ ID NOs: 14 and 15, and the nucleotide sequence of the probe is shown in SEQ ID NO: 16; The nucleotide sequences of the primers in the primer probe set 4 are shown in SEQ ID NOs: 17 and 18, and the nucleotide sequence of the probe is shown in SEQ ID NO: 19; The nucleotide sequences of the primers in the primer probe set 5 are shown in SEQ ID NOs: 20 and 21, and the nucleotide sequence of the probe is shown in SEQ ID NO: 22; The nucleotide sequences of the primers in the primer probe set 6 are shown in SEQ ID NOs: 23 and 24, and the nucleotide sequence of the probe is shown in SEQ ID NO: 25; The nucleotide sequences of the primers in the primer probe set 7 are shown in SEQ ID NOs: 26 and 27, and the nucleotide sequence of the probe is shown in SEQ ID NO: 28.

[0006] The present invention can efficiently detect Streptococcus agalactiae by selecting specific gene sequences to design primers and probes. cfb Genes and dits Campylobacter fetus gene sapB2 Genes and cadF gene, Staphylococcus aureus nuc Gene, Listeria monocytogenes hlyA Genes and Ureaplasma ure Gene.

[0007] Furthermore, in the primer probe group 1, the 5' end of the probe is labeled with a CY5 fluorescent group, and the 3' end is labeled with a BHQ-3 quencher group; in the primer probe group 2, the 5' end of the probe is labeled with a 6-FAM fluorescent group, and the 3' end is labeled with a BHQ1 quencher group; in the primer probe group 3, the 5' end of the probe is labeled with a ROX fluorescent group, and the 3' end is labeled with a BHQ2 quencher group; in the primer probe group 4, the 5' end of the probe is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ1 quencher group; in the primer probe group 5, the 5' end of the probe is labeled with a CY5 fluorescent group, and the 3' end is labeled with a BHQ-3 quencher group; in the primer probe group 6, the 5' end of the probe is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ1 quencher group; in the primer probe group 7, the 5' end of the probe is labeled with a 6-FAM fluorescent group, and the 3' end is labeled with a BHQ1 quencher group.

[0008] In a second aspect, the present invention provides the use of the primer-probe combination in the preparation of a kit for detecting pathogens; the pathogens include at least one of Streptococcus agalactiae, Campylobacter fetus, Staphylococcus aureus, Listeria monocytogenes and Ureaplasma.

[0009] Furthermore, the primer probe combination for detecting Streptococcus agalactiae comprises primer probe group 1 and / or primer probe group 2; The primer probe combination for detecting Campylobacter fetus comprises primer probe set 3 and / or primer probe set 4; The primer-probe combination for detecting Staphylococcus aureus contains primer-probe set 5; The primer-probe combination for detecting Listeria monocytogenes contains primer-probe set 6; The primer probe combination for detecting Ureaplasma ureaplasma contains primer probe set 7.

[0010] Furthermore, the detection of Streptococcus agalactiae cfb The primer-probe combination for the gene contains primer-probe set 1; Detection of Streptococcus agalactiae dits The primer-probe combination for the gene contains primer-probe set 2; Detection of Campylobacter fetus sapB2 The primer-probe combination for the gene contains primer-probe set 3; Detection of Campylobacter fetus cadF The primer-probe combination for the gene contains primer-probe set 4; Detection of Staphylococcus aureus nuc The primer-probe combination for the gene contains primer-probe set 5; Detection of Listeria monocytogenes hlyA The primer-probe combination for the gene contains primer-probe set 6; Detection of Ureaplasma ure The primer probe combination for the gene contains primer probe set 7.

[0011] Furthermore, the ureaplasma includes Ureaplasma microphylla and / or Ureaplasma urealyticum.

[0012] Furthermore, the pathogens include pathogens in vaginal secretions.

[0013] In a third aspect, the present invention provides a kit for detecting pathogens, wherein the kit contains the primer-probe combination.

[0014] Furthermore, the kit also contains PCR enzyme and PCR enzyme buffer.

[0015] Furthermore, the pathogen includes at least one of Streptococcus agalactiae, Campylobacter fetus, Staphylococcus aureus, Listeria monocytogenes and Ureaplasma.

[0016] Furthermore, the ureaplasma includes Ureaplasma microphylla and / or Ureaplasma urealyticum.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention is directed to Streptococcus agalactiae cfb Genes and dits Campylobacter fetus gene sapB2 Genes and cadF gene, Staphylococcus aureus nuc Gene, Listeria monocytogenes hlyA Genes and Ureaplasma ureWe designed a specific primer-probe combination and established a multiplex fluorescence quantitative PCR detection kit and method for screening common neonatal pathogens carried by mothers in the birth canal. It can detect seven pathogens at the same time with 100% specificity and over 85% sensitivity. The minimum detection limit reaches the femtogram level, which can provide timely feedback to the clinic for early intervention to avoid neonatal infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the positive detection curve of fluorescent quantitative PCR.

[0019] Figure 2 To detect Streptococcus agalactiae dits Standard curve of the gene.

[0020] Figure 3 To detect Streptococcus agalactiae cfb Standard curve of the gene.

[0021] Figure 4 To detect Campylobacter fetus sapB2 Standard curve of the gene.

[0022] Figure 5 To detect Campylobacter fetus cadF Standard curve of the gene.

[0023] Figure 6 To detect Staphylococcus aureus nuc Standard curve of the gene.

[0024] Figure 7 To detect Ureaplasma ure Standard curve of the gene.

[0025] Figure 8 To detect Listeria monocytogenes hlyA Standard curve of the gene. DETAILED DESCRIPTION

[0026] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0027] Example 1 Primer Design Comparative studies were performed using molecular evolutionary genetics analysis software (MEGA version 10.2.6, https: / / www.megasoftware.net) to identify nucleotide regions showing significant conservation.

[0028] To ensure the specificity of the probes and primers, the present invention downloaded 30 different bacteria dits Gene, cfb Gene, cadF Gene, sapB2 The DNA sequence of the gene was aligned using MEGA 10.2.6 software, avoiding the region of gene mutation and selecting the conserved region. Specific primers and probes were designed for the unique conserved region with the help of oligo7 (Molecular Biology Insights, https: / / www.oligo.net) and Beacon Designer 8.14 (PREMIER Biosoft, https: / / www.premierbiosoft.com). ure Gene, hlyA Genes and nuc The primer and probe combinations of the genes were consistent with the above method.

[0029] To assess potential primer-dimer formation and other parameters, the online tool Multiple Primer Analyzer (https: / / www.thermofisher.cn / cn / zh / home / brands / thermo-scientific / molecular-biology / molecular-biology-learning-center / molecular-biology-resource-library / thermo-scientific-web-tools / multiple-primer-analyzer.html) was used. This tool comprehensively evaluates primer and probe sets and selects the most effective combinations based on the highest ranking provided by the software's default settings.

[0030] As shown in Table 1, a method for detecting Streptococcus agalactiae ( cfb and dits ), Campylobacter fetus ( sapB2 and cadF ), Staphylococcus aureus ( nuc ), Listeria monocytogenes ( hlyA ) and Ureaplasma ( ure , including Ureaplasma microti and Ureaplasma urealyticum), and the target sequences corresponding to each primer and probe combination are shown in Table 2.

[0031] Streptococcus agalactiae ( cfb and dits ) and Campylobacter fetus ( sapB2 and cadF ) as group A, which will detect Staphylococcus aureus ( nuc ), Listeria monocytogenes ( hlyA) and Ureaplasma ( ure ) as group B, which is used to screen the vaginal secretions of pregnant women for the presence of neonatal pathogens, and provide timely feedback to the clinic for early intervention to avoid neonatal infection.

[0032] in, cfb The nucleotide sequence of the gene is shown in SEQ ID NO: 1. dits The nucleotide sequence of the gene is shown in SEQ ID NO: 2. sapB2 The nucleotide sequence of the gene is shown in SEQ ID NO: 3. cadF The nucleotide sequence of the gene is shown in SEQ ID NO: 4. nuc The nucleotide sequence of the gene is shown in SEQ ID NO: 5. hlyA The nucleotide sequence of the gene is shown in SEQ ID NO: 6, ure The nucleotide sequence of the gene is shown in SEQ ID NO:7.

[0033] Table 1

[0034] In Table 1, F: upstream primer; R: downstream primer; T: fluorescent probe.

[0035] The 5' and 3' ends of the fluorescent probe are also labeled with a fluorescent group and a quencher group, wherein, dtS Gene: 5' end labeled with 6-FAM fluorescent group, 3' end labeled with BHQ1 quencher group; cfb Gene: 5' end labeled with CY5 fluorescent group, 3' end labeled with BHQ-3 quencher group; cadF gene: 5' end labeled with VIC fluorescent group, 3' end labeled with BHQ1 quencher group; sapB2 gene: 5' end labeled with ROX fluorescent group, 3' end labeled with BHQ2 quencher group; ure gene: 5' end labeled with 6-FAM fluorescent group, 3' end labeled with BHQ1 quencher group; hlyA gene: 5' end labeled with VIC fluorescent group, 3' end labeled with BHQ1 quencher group; nuc gene: 5' end labeled with CY5 fluorescent group, 3' end labeled with BHQ-3 quencher group.

[0036] Table 2

[0037] Example 2 Detection limits and standard curves of different primers 1. Synthesis dtS Gene was serially diluted to obtain 10 ng / ml, 1 ng / ml, 100 pg / ml, 10 pg / ml, 1 pg / ml, 100 femtograms / ml, 10 femtograms / ml, and 1 femtogram / ml. dtSThe gene samples were used as templates for three multiplex fluorescence quantitative PCR tests.

[0038] The PCR reaction system consisted of 10 µL of the enzyme and mixture from the fluorescent quantitative PCR kit (TaqProbe 2X qPCR-Multiplex Master Mix, Catalog No. B630005-0005, Manufacturer: Sangon Biotech (Shanghai) Co., Ltd.), 0.8 µL of each upstream primer (400 nM), 0.8 µL of each downstream primer (400 nM), 0.4 µL of each fluorescent probe (200 nM), and 2 µL of template. Primers and probes were those listed in Table 1 for detecting Streptococcus agalactiae ( cfb and dits ) and Campylobacter fetus ( sapB2 and cadF ) primer and probe combinations.

[0039] The PCR reaction program was as follows: 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 40 s, for 40 cycles.

[0040] 2. Synthesis cfb Gene was serially diluted to obtain 10 ng / ml, 1 ng / ml, 100 pg / ml, 10 pg / ml, 1 pg / ml, 100 femtograms / ml, 10 femtograms / ml, 1 femtogram / ml, and 100 atg / ml. cfb The gene sample was used as a template for three multiplex fluorescence quantitative PCR assays. The PCR reaction system and PCR reaction procedure were the same as in step 1.

[0041] 3. Synthesis sapB2 Gene was serially diluted to obtain 10 ng / ml, 1 ng / ml, 100 pg / ml, 10 pg / ml, 1 pg / ml, 100 femtograms / ml, 10 femtograms / ml, and 1 femtogram / ml. sapB2 The gene sample was used as a template for three multiplex fluorescence quantitative PCR assays. The PCR reaction system and PCR reaction procedure were the same as in step 1.

[0042] 4. Synthesis cadF Gene was serially diluted to obtain 10 ng / ml, 1 ng / ml, 100 pg / ml, 10 pg / ml, 1 pg / ml, 100 femtograms / ml, 10 femtograms / ml, and 1 femtogram / ml. cadF The gene sample was used as a template for three multiplex fluorescence quantitative PCR assays. The PCR reaction system and PCR reaction procedure were the same as in step 1.

[0043] 5. Synthesis nucThe nuc gene was serially diluted to obtain 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, 1 pg / mL, 100 femtograms / mL, 10 femtograms / mL, 1 femtogram / mL, and 100 atg / mL nuc gene samples. These samples were used as templates for three multiplex fluorescence quantitative PCR assays. The PCR reaction system and PCR reaction procedure were the same as in step 1, with the primers and probes for detecting Staphylococcus aureus (Table 1) as described. nuc ), Listeria monocytogenes ( hlyA ) and Ureaplasma ( ure ) primer and probe combinations.

[0044] 6. Synthesis ure Gene was serially diluted to obtain 10 ng / ml, 1 ng / ml, 100 pg / ml, 10 pg / ml, 1 pg / ml, 100 femtograms / ml, 10 femtograms / ml, 1 femtogram / ml, and 100 atg / ml. ure The gene sample was used as a template for three multiplex fluorescence quantitative PCR assays. The PCR reaction system and PCR reaction procedure were the same as in step 5.

[0045] 7. Synthesis hlyA Gene was serially diluted to obtain 10 ng / ml, 1 ng / ml, 100 pg / ml, 10 pg / ml, 1 pg / ml, 100 femtograms / ml, 10 femtograms / ml, 1 femtogram / ml, and 100 atg / ml. hlyA The gene sample was used as a template for three multiplex fluorescence quantitative PCR assays. The PCR reaction system and PCR reaction procedure were the same as in step 5.

[0046] like Figure 1 As shown, the results of 3 tests show dtS The minimum detection limit of the primers for the gene was 14.33±0.23 femtograms / ml. Figure 2 As shown, for detection dtS The standard curve of the gene can be used for the detection of routine samples.

[0047] The results of 3 tests showed cfb The minimum detection limit of the primers for the gene was 1.17±0.57 femtograms / ml. Figure 3 As shown, for detection cfb The standard curve of the gene can be used for the detection of routine samples.

[0048] The results of 3 tests showed sapB2 The minimum detection limit of the primers for the gene was 32.23±2.82 femtograms / ml. Figure 4 As shown, for detection sapB2 The standard curve of the gene can be used for the detection of routine samples.

[0049] The results of 3 tests showed cadF The minimum detection limit of the primers for the gene was 32.17±7.26 femtograms / ml. Figure 5 As shown, for detection cadF The standard curve of the gene can be used for the detection of routine samples.

[0050] The results of 3 tests showed nuc The minimum detection limit of the primers for the gene was 1.29±0.004 femtograms / ml. Figure 6 As shown, for detection nuc The standard curve of the gene can be used for the detection of routine samples.

[0051] The results of 3 tests showed ure The minimum detection limit of the primers for the gene was 3.56±0.29 femtograms / ml. Figure 7 As shown, for detection ure The standard curve of the gene can be used for the detection of routine samples.

[0052] The results of 3 tests showed hlyA The minimum detection limit of the primers for the gene was 1.29±0.12 femtograms / ml. Figure 8 As shown, for detection hlyA The standard curve of the gene can be used for the detection of routine samples.

[0053] Example 3 Specificity Detection Nucleic acids of 165 bacterial strains, including 15 strains of Escherichia coli, 20 strains of Klebsiella pneumoniae, 30 strains of Candida, 25 strains of Gardnerella vaginalis, 30 strains of Lactobacillus, 15 strains of Enterococcus, and 30 strains of Staphylococcus epidermidis, were extracted according to conventional laboratory methods or a nucleic acid extraction kit and used as templates for detection using the detection method of Example 2.

[0054] The test results were all negative with a specificity of 100%, indicating that the primer probe designed by the present invention would not produce nonspecific amplification with common pathogens, leading to false positives.

[0055] Example 4 Actual sample detection The nucleic acid of the vaginal secretion sample of the parturient is extracted according to conventional laboratory methods or a nucleic acid extraction kit as a template, and the detection method of Example 2 is used for detection.

[0056] A total of 15 specimens were found to contain Streptococcus agalactiae, 4 specimens contained Ureaplasma, and 12 specimens contained Staphylococcus aureus. Listeria monocytogenes and Campylobacter fetus were not detected. Compared with traditional culture methods, the detection rates of Streptococcus agalactiae were 86.7%, Ureaplasma were 90% (9 / 10), and Staphylococcus aureus were 85.71% (12 / 14). This indicates that the detection method developed by this invention can be used for routine clinical screening of pregnant women and reduce the risk of neonatal infection.

[0057] Example 5 A kit for detecting pathogens 1. Composition The primer-probe combinations shown in Table 1 will detect Streptococcus agalactiae ( cfb and dits ) and Campylobacter fetus ( sapB2 and cadF ) as group A, which will detect Staphylococcus aureus ( nuc ), Listeria monocytogenes ( hlyA ) and Ureaplasma ( ure ) are used as group B.

[0058] Conventional PCR enzymes and PCR enzyme buffers can be used and are also commercially available.

[0059] Positive control: genomic DNA of Streptococcus agalactiae, Campylobacter fetus, Staphylococcus aureus, Listeria monocytogenes or Ureaplasma, the recommended concentration of each genomic DNA is 10 6 Femtograms / mL, higher concentrations are possible. Negative control: deionized water.

[0060] 2. Usage 1. Extract nucleic acid from vaginal secretion specimens of parturient women as a template using routine laboratory methods or a nucleic acid extraction kit.

[0061] 2. Perform PCR reaction on the template, positive control and negative control in step 1 respectively.

[0062] The PCR reaction system in tube A is as follows: 10 µL of the enzyme and mixture of the fluorescent quantitative PCR kit (TaqProbe 2X qPCR-Multiplex Master Mix, Catalog No.: B630005-0005, Manufacturer: Sangon Biotech (Shanghai) Co., Ltd.), 0.8 µL of each upstream primer (400 nM) in group A, 0.8 µL of each downstream primer (400 nM) in group A, 0.4 µL of each fluorescent probe (200 nM) in group A, and 2 µL of template.

[0063] The PCR reaction system in tube B is as follows: 10 µL of the enzyme and mixture of the fluorescent quantitative PCR kit (TaqProbe 2X qPCR-Multiplex Master Mix, Catalog No.: B630005-0005, Manufacturer: Sangon Biotech (Shanghai) Co., Ltd.), 0.8 µL of each upstream primer (400 nM) in group B, 0.8 µL of each downstream primer (400 nM) in group B, 0.4 µL of each fluorescent probe (200 nM) in group B, and 2 µL of template.

[0064] The PCR reaction program was as follows: 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 40 s, for 40 cycles.

[0065] 3. Determination method When the amplification result shows positive amplification, that is, the CT value is displayed and the corresponding calculated concentration is obtained, it is determined to be a positive result. When there is no positive amplification, that is, there is no CT value and calculated concentration, it is determined to be a negative result. Note: Each time an amplification experiment is performed, the genomic DNA of the five pathogens should be diluted to 4 gradient concentrations as a standard (for example, 10 8 , 10 7 , 10 6 , 10 5 , femtogram / ml) to draw a standard curve, and at the same time, use positive and negative controls to monitor whether there are any problems with the entire operation process and reagents.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A primer-probe combination, characterized in that: The primer-probe combination contains any one of primer-probe groups 1 to 7; The nucleotide sequences of the primers in the primer-probe set 1 are shown in SEQ ID NOs: 8 and 9, and the nucleotide sequence of the probe is shown in SEQ ID NO: 10; The nucleotide sequences of the primers in the primer probe set 2 are shown in SEQ ID NOs: 11 and 12, and the nucleotide sequence of the probe is shown in SEQ ID NO: 13; The nucleotide sequences of the primers in the primer probe set 3 are shown in SEQ ID NOs: 14 and 15, and the nucleotide sequence of the probe is shown in SEQ ID NO: 16; The nucleotide sequences of the primers in the primer probe set 4 are shown in SEQ ID NOs: 17 and 18, and the nucleotide sequence of the probe is shown in SEQ ID NO: 19; The nucleotide sequences of the primers in the primer probe set 5 are shown in SEQ ID NOs: 20 and 21, and the nucleotide sequence of the probe is shown in SEQ ID NO: 22; The nucleotide sequences of the primers in the primer probe set 6 are shown in SEQ ID NOs: 23 and 24, and the nucleotide sequence of the probe is shown in SEQ ID NO: 25; The nucleotide sequences of the primers in the primer probe set 7 are shown in SEQ ID NOs: 26 and 27, and the nucleotide sequence of the probe is shown in SEQ ID NO:

28.

2. The primer-probe combination according to claim 1, wherein In the primer probe group 1, the 5' end of the probe is labeled with a CY5 fluorescent group, and the 3' end is labeled with a BHQ-3 quencher group; in the primer probe group 2, the 5' end of the probe is labeled with a 6-FAM fluorescent group, and the 3' end is labeled with a BHQ1 quencher group; in the primer probe group 3, the 5' end of the probe is labeled with a ROX fluorescent group, and the 3' end is labeled with a BHQ2 quencher group; in the primer probe group 4, the 5' end of the probe is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ1 quencher group; in the primer probe group 5, the 5' end of the probe is labeled with a CY5 fluorescent group, and the 3' end is labeled with a BHQ-3 quencher group; in the primer probe group 6, the 5' end of the probe is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ1 quencher group; in the primer probe group 7, the 5' end of the probe is labeled with a 6-FAM fluorescent group, and the 3' end is labeled with a BHQ1 quencher group.

3. Use of the primer-probe combination according to claim 1 or 2 in the preparation of a kit for detecting pathogens; the pathogens include at least one of Streptococcus agalactiae, Campylobacter fetus, Staphylococcus aureus, Listeria monocytogenes and Ureaplasma.

4. The use according to claim 3, characterized in that The primer probe combination for detecting Streptococcus agalactiae comprises primer probe set 1 and / or primer probe set 2; The primer probe combination for detecting Campylobacter fetus comprises primer probe set 3 and / or primer probe set 4; The primer-probe combination for detecting Staphylococcus aureus contains primer-probe set 5; The primer-probe combination for detecting Listeria monocytogenes contains primer-probe set 6; The primer probe combination for detecting Ureaplasma ureaplasma contains primer probe set 7.

5. The use according to claim 4, characterized in that Detection of Streptococcus agalactiae cfb The primer-probe combination for the gene contains primer-probe set 1; Detection of Streptococcus agalactiae dits The primer-probe combination for the gene contains primer-probe set 2; Detection of Campylobacter fetus sapB2 The primer-probe combination for the gene contains primer-probe set 3; Detection of Campylobacter fetus cadF The primer-probe combination for the gene contains primer-probe set 4; Detection of Staphylococcus aureus nuc The primer-probe combination for the gene contains primer-probe set 5; Detection of Listeria monocytogenes hlyA The primer-probe combination for the gene contains primer-probe set 6; Detection of Ureaplasma ure The primer probe combination for the gene contains primer probe set 7.

6. The use according to claim 3, characterized in that The ureaplasma includes Ureaplasma microti and / or Ureaplasma urealyticum.

7. The use according to claim 3, wherein: The pathogens include pathogens in vaginal secretions.

8. A kit for detecting pathogens, characterized in that: The kit contains the primer-probe combination according to claim 1 or 2.

9. The kit according to claim 8, wherein The pathogens include at least one of Streptococcus agalactiae, Campylobacter fetus, Staphylococcus aureus, Listeria monocytogenes and Ureaplasma.

10. The kit according to claim 9, wherein The ureaplasma includes Ureaplasma microti and / or Ureaplasma urealyticum.

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