Primer and probe combination and kit for detecting exogenous pathogens of gynecological diseases
By providing a combination of primers and probes for exogenous pathogens of gynecological diseases, combined with multiple qPCR methods, the ability to detect multiple pathogens simultaneously is achieved, solving the problems of low detection efficiency and high cost in the prior art, and achieving efficient, accurate and economical pathogen diagnosis.
Patent Information
- Application Number
- CN202510312890.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-17
- Publication Date
- 2025-05-06
AI Technical Summary
The prior art has low sensitivity and poor specificity when detecting exogenous pathogens of gynecological diseases. Multiple detections require multiple detections, which is time-consuming and labor-intensive and costly.
It provides a combination of primers and probes for detecting trichomonas vaginalis, Mycoplasma humanoid, Ureaplasma urea and Candida leucidum, combined with multiple qPCR methods to achieve detection of multiple targets at one time.
It improves detection efficiency, accurate detection results, reduces detection costs, can quickly complete pathogen diagnosis, and has a high level of specificity and sensitivity.
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Figure CN119932214A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a primer and probe combination and a kit for detecting exogenous pathogens of gynecological diseases, and belongs to the field of biotechnology. Background Art
[0002] The vagina is a typical micro-ecosystem, with different types and numbers of microorganisms, which together maintain a vaginal microenvironment dominated by lactic acid bacteria. When the balance of the vaginal microenvironment is disrupted, it will cause lower genital tract infections and further cause gynecological diseases. An important reason for disrupting the balance of the vaginal microenvironment is the invasion of exogenous pathogens. At present, exogenous pathogens of gynecological diseases include: Trichomonas vaginalis, Chlamydia trachomatis, Toxoplasma gondii, Candida albicans, Mycoplasma hominis, Ureaplasma urealyticum, Mycobacterium tuberculosis, cytomegalovirus, etc. Among these exogenous pathogens, Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Candida albicans (CA) and Ureaplasma urealyticum (UU) often cause mixed infections.
[0003] Infections with Trichomonas vaginalis, Candida albicans, Mycoplasma hominis and Ureaplasma urealyticum can cause vaginitis, urethritis and cervicitis in women, and are associated with the risk of pelvic inflammatory disease, infertility, cervical tumors and poor delivery outcomes. Trichomonas vaginalis (TV) is a unicellular, flagellated, microaerophilic protozoan. Humans are the only natural host. Trichomoniasis caused by infection is the most common curable sexually transmitted infection (STI). Candida albicans (CA), also known as Candida albicans or Candida albicans. Candida is the most common fungal group in the vagina, mainly Candida albicans, and is a conditional pathogen. When the vaginal microecology is out of balance (pH value changes or antibiotic use, changes in immune function, etc.), the vaginal microenvironment is suitable for the reproduction and growth of Candida, which will cause vulvovaginal candidiasis (VVC). Symptoms are usually characterized by increased vaginal discharge, frequent urination, painful urination, vulvar itching, and burning. 75%-80% of women will be infected with VVC at least once in their lives, and up to 9% of patients will relapse and develop recurrent VVC. VVC can also induce cervicitis and pelvic inflammatory disease. Repeated infection can change the pH of the vagina and lead to infertility. Infection during pregnancy may cause chorioamnionitis, amniotic fluid infection, premature rupture of membranes, premature birth, and endometrial infection after cesarean section or vaginal delivery, which seriously affects women's health. Mycoplasma hominis (MH) and Ureaplasma urealyticum (UU) are the smallest prokaryotic cell-type microorganisms that have no cell wall, can pass through bacterial filters, and can grow in inanimate culture media. They colonize the human reproductive tract. Due to the lack of cell walls, they are resistant to β-lactams and all antibiotics targeting the cell wall. They are mainly transmitted through sexual transmission, and can also be transmitted vertically or through transplanted organs. They can cause asymptomatic, long-term and chronic urogenital tract infections, such as non-gonococcal urethritis (NGU), prostatitis, pelvic inflammatory disease, cervicitis and bacterial vaginitis, increase the risk of ectopic pregnancy and neonatal diseases, and can affect human fertility, leading to infertility, etc.
[0004] At present, the commonly used methods for detecting these four exogenous pathogens in clinical practice include smear microscopy, culture, and immunological methods, but these methods have low sensitivity, poor specificity, or take a long time to detect; real-time fluorescence quantitative PCR (qPCR) has advantages such as good sensitivity and specificity, but can only detect one target in a reaction tube. If it is used to detect multiple indicators, multiple tests are required, which is not only time-consuming and labor-intensive, but also sharply increases the cost. Multiplex qPCR is based on qPCR and can detect multiple targets in a single reaction tube at one time, shortening the detection time and greatly reducing the detection cost. It has its unique advantages and high practical value in the rapid diagnosis of clinical infection indicators, but there are currently no reports on the simultaneous detection of these four exogenous pathogens of gynecological diseases using multiplex qPCR. Summary of the invention
[0005] In view of some deficiencies in the prior art, the present invention provides a primer and probe combination and a kit for detecting exogenous pathogens of gynecological diseases, and in particular provides a primer and probe combination and a kit for simultaneously detecting multiple exogenous pathogens of gynecological diseases, wherein the exogenous pathogens of gynecological diseases include Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and Candida albicans (CA).
[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:
[0007] The present invention firstly simultaneously detects a plurality of exogenous pathogens of gynecological diseases using a primer and probe combination, wherein the combination includes specific primers and probes for detecting Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, and Candida albicans;
[0008] Among them, the specific primers and probes for detecting Trichomonas vaginalis include primers with nucleotide sequences shown in SEQ ID Nos: 1-2 and probes with nucleotide sequences shown in SEQ ID Nos: 3; the specific primers and probes for detecting Mycoplasma hominis include primers with nucleotide sequences shown in SEQ ID Nos: 4-5 and probes with nucleotide sequences shown in SEQ ID Nos: 6; the specific primers and probes for detecting Ureaplasma urealyticum include primers with nucleotide sequences shown in SEQ ID Nos: 7-8 and probes with nucleotide sequences shown in SEQ ID Nos: 9; the specific primers and probes for detecting Candida albicans include primers with nucleotide sequences shown in SEQ ID Nos: 10-11 and probes with nucleotide sequences shown in SEQ ID Nos: 12.
[0009] Furthermore, the 5' end of the probe is labeled with a reporter fluorescent group, and the 3' end is labeled with a fluorescent quencher group.
[0010] Furthermore, the reporter fluorescent groups include 6-FAM, ROX, CY5, and VIC, and the fluorescent quenching groups include BHQ1, BHQ2, and BHQ3.
[0011] Further preferably, the probe for detecting Trichomonas vaginalis has a reporter fluorescent group 6-FAM at the 5' end and a fluorescent quencher group BHQ1 at the 3' end; the probe for detecting Mycoplasma hominis has a reporter fluorescent group ROX at the 5' end and a fluorescent quencher group BHQ2 at the 3' end; the probe for detecting Ureaplasma urealyticum has a reporter fluorescent group CY5 at the 5' end and a fluorescent quencher group BHQ3 at the 3' end; the probe for detecting Candida albicans has a reporter fluorescent group VIC at the 5' end and a fluorescent quencher group BHQ1 at the 3' end.
[0012] The present invention also provides the use of the primer and probe combination in preparing a reagent or a kit for simultaneously detecting multiple exogenous pathogens of gynecological diseases.
[0013] The present invention also provides a kit for simultaneously detecting multiple exogenous pathogens of gynecological diseases, and the kit comprises the primer and probe combination.
[0014] Furthermore, the test sample of the kit includes a vaginal swab.
[0015] The kit also includes a sample DNA extraction reagent, and the kit also includes a multiplex qPCR detection reagent, and the multiplex qPCR detection reagent includes Premix Ex Taq (2X) and enzyme-free water.
[0016] The kit also includes a negative control and a positive control. The negative control includes enzyme-free water, and the positive control includes a plasmid standard containing specific sequences of Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, and Candida albicans.
[0017] Interpretation of positive test results: The amplification curve shows a standard "S" shape without abnormal fluctuations. A pathogen Ct value ≤35.0 can be judged as positive. If the Ct value is greater than 35.0, a single real-time fluorescence quantitative PCR verification is required for the pathogen.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] (1) The primer and probe combination provided by the present invention for simultaneously detecting four pathogens, namely Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and Candida albicans (CA), combined with the multiplex qPCR method, has high detection efficiency and accurate detection results, and can quickly complete pathogen diagnosis at low cost.
[0020] Further specificity evaluation of the quadruple qPCR (mqPCR) method for Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and Candida albicans (CA) showed that there was no cross-reaction between the pathogen and other pathogens, and the specificity of each pathogen was good; sensitivity evaluation found that the sensitivity of Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and Candida albicans (CA) all reached 10 copies / μL, with a high sensitivity; the accuracy of mqPCR was evaluated by testing 10 vaginal swab samples, and the results showed that the mqPCR method had good accuracy.
[0021] (2) The present invention is based on a real-time fluorescence quantitative PCR technology platform and provides a primer and probe combination and a kit that can simultaneously detect multiple exogenous pathogens of gynecological diseases, perform qualitative detection of pathogen-specific target genes, and can detect four pathogens at a time. It has the characteristics of rapidity, specificity, and economy, greatly reducing the detection cost of each sample. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is the single qPCR specificity test result of TV in Example 2.
[0023] Figure 2 This is the single qPCR specificity test result of MH in Example 2.
[0024] Figure 3 This is the single qPCR specificity test result of UU in Example 2.
[0025] Figure 4 This is the single qPCR specificity test result of CA in Example 2.
[0026] Figure 5 These are the results of the quadruple qPCR specificity test of TV, MH, UU, and CA in Example 2.
[0027] Figure 6 This is the result of the quadruple qPCR sensitivity test of TV in Example 2.
[0028] Figure 7 This is the result of the quadruple qPCR sensitivity test of MH in Example 2.
[0029] Figure 8 These are the results of the quadruple qPCR sensitivity test of UU in Example 2.
[0030] Fig. 9 The results of the quadruple qPCR sensitivity test of CA in Example 2. DETAILED DESCRIPTION
[0031] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.
[0032] In the embodiments of the present invention, those that are not described in detail are all completed by conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail.
[0033] The Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Neisseria gonorrhoeae, and Streptococcus agalactiae described in the examples are obtained by clinical isolation and identification through conventional methods; they can also be cultured using conventional culture methods; or they can be purchased.
[0034] Example 1: Design of primers and probes for detecting Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and Candida albicans (CA)
[0035] Primers and probes were designed based on the exogenous pathogen gene reference sequences in the NCBI (https: / / www.ncbi.nlm.nih.gov / ) website, including: Trichomonas vaginalis beta-tubulin (btub1) encoding gene (accession number: L05468.1), Mycoplasma hominis 16S ribosomal RNA encoding gene (accession number: M96660.1), Ureaplasma urealyticum WJSL encoding gene (accession number: CP062067.1), Candida albicans L757 mitochondrion encoding gene (accession number: NC_018046.1).
[0036] When using Beacon Designer 8 software to design primers and probes, the following conditions must be met for screening:
[0037] (1) The length of primers and probes (L) should be between 18 and 30 bp.
[0038] (2) The primer Tm value is about 8-10°C lower than the probe Tm value.
[0039] (3) The GC content of primers and probes is generally not less than 40%.
[0040] (4) The 3' end of the primer should not contain more than three consecutive G or C sequences.
[0041] (5) The Tm value variation between primers is no more than 2°C.
[0042] (6) The length of the amplified product is controlled between 70 and 150 bp.
[0043] Use the BLAST search function on the NCBI website to compare and evaluate the nucleotide sequences of the primers and probes that have been preliminarily designed, and select primers and probe sequences with high specificity;
[0044] The nucleotide sequences of the specific primers and probes targeting Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and Candida albicans (CA) are shown in SEQ ID No: 1 to SEQ ID No: 12, as shown in the following table:
[0045] Table 1. Specific primers and probe sequences for detecting various pathogens
[0046] Serial number Primer and probe name sequence serial number 1 TV upstream primer 5'-TCAACAAGCTCCGTGAAGAG-3' SEQ ID No: 1 2 TV downstream primer 5'-CGGAGAGTGTGCAGTTGTAT-3' SEQ ID No: 2 3 TV Taqman Probes 5'6-FAM-CTCTCCACATACTCCATCGTCCCATCT-BHQ1-3' SEQ ID No: 3 4 MH upstream primer 5'-GCATGGTTCCGTTGTGAAAG-3' SEQ ID No: 4 5 MH downstream primer 5'-CATAGTCTTGGTGGGCCATTA-3' SEQ ID No: 5 6 MH Taqman Probe 5'ROX-TCACCAACTAACTAATGTTCCGCACCC-BHQ2-3' SEQ ID No: 6 7 UU upstream primer 5'-AGTCCTAGCGAAGCAATAGC-3' SEQ ID No: 7 8 UU downstream primer 5'-GGATCAATTACATCAGTCCAAGC-3' SEQ ID No: 8 9 UU Taqman Probes 5'CY5-AGCTGACGACTTAACAGACCCTGCG-BHQ3-3' SEQ ID No: 9 10 CA upstream primer 5'-GTGGTGTGCCAGCAGTAG-3' SEQ ID No: 10 11 CA downstream primer 5'-GCCTACGGGTCCTTTATGC-3' SEQ ID No: 11 12 CA Taqman Probes 5'VIC-CATACACCCAGAGCGAGCGTTGATC-BHQ1-3' SEQ ID No: 12
[0047] The reporter fluorescent group at the 5' end of the Trichomonas vaginalis TaqMan probe (SEQ ID No: 3) of the present invention is 6-FAM, and the fluorescent quenching group at the 3' end is BHQ1; the reporter fluorescent group at the 5' end of the Mycoplasma hominis TaqMan probe (SEQ ID No: 6) is ROX, and the fluorescent quenching group at the 3' end is BHQ2; the reporter fluorescent group at the 5' end of the Ureaplasma urealyticum TaqMan probe (SEQ ID No: 9) is CY5, and the fluorescent quenching group at the 3' end is BHQ3; the reporter fluorescent group at the 5' end of the Candida albicans TaqMan probe (SEQ ID No: 12) is VIC, and the fluorescent quenching group at the 3' end is BHQ1.
[0048] The specific sequences of four exogenous pathogens, Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and Candida albicans (CA) (NCBI accession number: L05468.1, accession number: M96660.1, accession number: CP062067.1, accession number: NC_018046.1) were connected to the pUC57 vector to synthesize plasmid standards. The plasmid construction was completed by Sangon Biotech (Shanghai) Co., Ltd.; the concentration was measured by ultraviolet spectrophotometer, and the copy number of the plasmid was calculated according to the length and concentration of each plasmid standard (see Table 2). The calculation formula is as follows:
[0049]
[0050] Table 2. Plasmid copy numbers of various exogenous pathogens
[0051] Pathogens Plasmid concentration (ng / μL) Plasmid size (bp) Plasmid copy number (copies / μL) Trichomonas vaginalis (TV) 45.23 2821 <![CDATA[1.46*10 10 ]]> Mycoplasma hominis (MH) 45.69 2807 <![CDATA[1.49*10 10 ]]> Ureaplasma urealyticum (UU) 46.03 2838 <![CDATA[1.51*10 10 ]]> Candida albicans (CA) 46.61 2783 <![CDATA[1.50*10 10 ]]>
[0052] The plasmid standards in Table 2 were diluted in a 10-fold gradient, with a total of six gradients, namely, 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10copies / μL level.
[0053] Example 2: qPCR amplification and specificity and sensitivity test
[0054] (1) Single fluorescence quantitative PCR:
[0055] Table 3. Single fluorescence quantitative PCR reaction system
[0056]
[0057] The amplification reaction program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 61°C for 35 s, and 40 cycles, with fluorescence signals collected during the annealing and extension phases of each cycle.
[0058] Premix Ex Taq (2×) (Probe qPCR) (TaKaRa) was added with the specific primers and probes for TV, MH, UU, and CA designed in Example 1 to prepare a single fluorescent quantitative PCR reaction system for TV, MH, UU, and CA. 3 The single fluorescence quantitative PCR reaction system of TV, MH, UU and CA was used as templates, and the test results were as follows: Figure 1-4 As shown, the plasmid templates of TV, MH, UU, CA and the corresponding single fluorescence quantitative PCR detection results of TV, MH, UU, CA all have specific amplification curves; no amplification curves appear when a mixed plasmid without target bacteria (such as MH, UU, CA but without TV) is amplified in a single fluorescence quantitative PCR reaction system containing primers and probes of target bacteria (such as TV). This shows that the specific primers and probes for TV, MH, UU, CA designed in Example 1 can detect the synthesized plasmid templates of TV, MH, UU, CA separately and specifically.
[0059] (2) Multiplex qPCR specificity test:
[0060] Table 4. Multiplex fluorescence quantitative PCR reaction system
[0061] Reagents Usage Premix Ex Taq(2×)(Probe qPCR) 10.0μL TV PCR Forward Primer (10μM) 0.15μL TV PCR Reverse Primer (10μM) 0.15μL TV Probe (10μM) 0.3μL MH PCR Forward Primer (10μM) 0.15μL MH PCR Reverse Primer (10μM) 0.15μL MH Probe (10μM) 0.3μL UU PCR Forward Primer (10μM) 0.3μL UU PCR Reverse Primer (10μM) 0.3μL UU Probe (10μM) 0.6μL CA PCR Forward Primer (10μM) 0.15μL CAPCR Reverse Primer (10μM) 0.15μL CA Probe (10μM) 0.3μL template 2μL DEPC water 5μL Total 20μL
[0062] The amplification reaction program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 61°C for 35 s, and 40 cycles, with fluorescence signals collected during the annealing and extension phases of each cycle.
[0063] Premix Ex Taq (2×) (Probe qPCR) (TaKaRa) was used to add TV, MH, UU, and CA specific primers and probes to prepare TV, MH, UU, and CA quadruple fluorescence quantitative PCR reaction system. 3 A mixture of 100 copies / μL TV, MH, UU, and CA plasmid standard products was used as a template, and a quadruple PCR reaction system containing TV, MH, UU, and CA primers was added; and a mixture of DNA extracted from clinical isolates of Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Neisseria gonorrhoeae, and Streptococcus agalactiae was used as a template and added to the quadruple reaction system as a negative control.
[0064] According to the interpretation criteria of the positive test results: the amplification curve is a standard "S" shape without abnormal fluctuations, and the pathogen Ct value ≤ 35.0 can be judged as positive. If the Ct value is greater than 35.0, a single real-time fluorescence quantitative PCR verification is required for the pathogen. Figure 5 As shown, it can be seen from the results that there is no cross reaction between the pathogens in each group, indicating that the multiplex fluorescence quantitative PCR constructed by the present invention has good specificity.
[0065] (3) Multiplex qPCR sensitivity test:
[0066] The gradient 10 set in Example 2 was quantified by multiple qPCR. 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 copies / μL of plasmid standards were tested to determine the lowest plasmid concentration that can be detected by the multiplex qPCR detection method. The results are as follows Figure 6-9 As shown in the figure, the lower limits of detection of TV, MH, CA and UU all reached 10 copies / μL.
[0067] Example 3: Detection of vaginal swab
[0068] Vaginal swabs were routinely collected from patients with clinical vaginitis.
[0069] For genomic DNA extraction, the sample is a vaginal swab; the vaginal swab is placed in a 2 mL centrifuge tube filled with normal saline, shaken thoroughly, and the nucleic acid is extracted using the Kangwei Century Universal Column Genome Extraction Kit according to the instructions.
[0070] Vaginal swab samples from 10 patients with positive wet smears for Trichomonas vaginalis were collected, and the DNA of the vaginal swab samples was detected using the quadruple qPCR detection reaction system provided in Example 2. At the same time, ordinary PCR was used for detection, and the test results were recorded. The results are as follows:
[0071] Table 5. Results of quadruple qPCR and conventional PCR
[0072] Test samples Quadruple qPCR results Normal PCR results 1 TV, MH, UU TV, MH, UU 2 TV, MH, UU TV, MH, UU 3 TV, UU, CA TV, UU, CA 4 TV TV 5 TV, MH TV, MH 6 TV, MH TV, MH 7 TV, MH, CA TV, MH, CA 8 TV, MH TV, MH 9 TV, MH, UU TV, MH, UU 10 TV, CA TV, CA
[0073] As can be seen from the above table, the quadruple qPCR detection method of TV, MH, CA and UU constructed by the present invention has similar detection results compared with the conventional PCR detection method, and the multiplex qPCR detection method of the present invention is more rapid.
[0074] The amplified products of the common PCR in Table 5 were subjected to first generation sequencing (Sanger sequencing), and the sequencing results were compared by Blast on the NCBI website to determine the type of pathogen, which was consistent with the results of the common PCR determination, further indicating that the method of the present invention has high accuracy.
[0075] The present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A primer and probe combination for simultaneously detecting multiple exogenous pathogens of gynecological diseases, characterized in that: The combination includes specific primers and probes for detecting Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, and Candida albicans; The specific primers and probes for detecting Trichomonas vaginalis include primers with nucleotide sequences shown in SEQ ID No: 1-2 and probes with nucleotide sequences shown in SEQ ID No: 3; The specific primers and probes for detecting Mycoplasma hominis include primers with nucleotide sequences shown in SEQ ID No: 4-5 and probes with nucleotide sequences shown in SEQ ID No: 6; The specific primers and probes for detecting Ureaplasma urealyticum include primers with nucleotide sequences shown in SEQ ID No: 7-8 and probes with nucleotide sequences shown in SEQ ID No: 9; The specific primers and probes for detecting Candida albicans include primers with nucleotide sequences shown in SEQ ID Nos: 10-11 and a probe with a nucleotide sequence shown in SEQ ID No:
12.
2. The primer and probe combination according to claim 1, characterized in that: The 5' end of the probe is labeled with a reporter fluorescent group, and the 3' end is labeled with a fluorescent quencher group.
3. The primer and probe combination according to claim 2, characterized in that: The reporter fluorescent groups include 6-FAM, ROX, CY5, and VIC, and the fluorescent quenching groups include BHQ1, BHQ2, and BHQ3.
4. The primer and probe combination according to claim 2, characterized in that: The probe for detecting Trichomonas vaginalis has a reporter fluorescent group 6-FAM at the 5' end and a fluorescent quencher group BHQ1 at the 3' end; The probe for detecting Mycoplasma hominis has a reporter fluorescent group ROX at the 5' end and a fluorescent quencher group BHQ2 at the 3' end; The 5' end of the probe for detecting Ureaplasma urealyticum has a reporter fluorescent group CY5, and the 3' end has a fluorescent quenching group BHQ3; The probe for detecting Candida albicans has a reporter fluorescent group VIC at the 5' end and a fluorescent quencher group BHQ1 at the 3' end.
5. Use of the primer and probe combination according to any one of claims 1 to 4 in the preparation of a reagent or kit for simultaneously detecting multiple exogenous pathogens of gynecological diseases.
6. A kit for simultaneously detecting multiple exogenous pathogens of gynecological diseases, characterized in that: The kit comprises the primer and probe combination according to any one of claims 1 to 4.
7. The kit according to claim 6, characterized in that The test sample of the kit includes a vaginal swab.
8. The kit according to claim 6, characterized in that The kit also includes a sample DNA extraction reagent.
9. The kit according to claim 6, characterized in that The kit also includes multiplex qPCR detection reagents, which include Premix Ex Taq (2X) and enzyme-free water.
10. The kit according to claim 6, characterized in that The kit also includes a negative control and a positive control, wherein the negative control includes enzyme-free water; The positive control includes plasmid standards containing specific sequences of Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, and Candida albicans.