A primer probe combination, kit and application for detecting pathogenic microorganisms in the genital tract

Abstract

The application discloses a primer probe combination, a kit and application for detecting pathogenic microorganisms in a genital tract, wherein the primer probe combination comprises primer probe sequences shown in SEQ ID NO. 1-SEQ ID NO. 96, the pathogenic microorganisms in the genital tract include 27 pathogenic microorganisms which can cause diseases in the genital tract, such as Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Candida albicans, Candida glabrata, Candida tropicalis, Candida auris, Candida krusei, Candida dubliniensis and the like, and four common vaginal lactobacilli, such as Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus iners, and further include a human internal standard. Based on the fluorescence quantitative PCR technology, the 27 pathogenic microorganisms which can cause diseases in the genital tract and the four common vaginal lactobacilli can be simultaneously detected in an in-vitro sample, the main pathogenic microorganisms of the genital tract infection can be covered, the evaluation of the vaginal ecological environment can be satisfied, the detection time is shortened to about 40 minutes, and the specificity and the sensitivity are good.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a primer probe combination for detecting pathogenic microorganisms in the genital tract, a kit and application. BACKGROUND

[0002] Genital tract infection is a global social and public health problem, with a high incidence rate worldwide, especially in sexually active people, and the incidence rate in women is significantly higher than that in men. Diseases caused by genital tract infection usually include bacterial vaginosis (BV), aerobic vaginitis (AV), vulvovaginal candidiasis (VVC), trichomonas vaginitis (TV) and sexually transmitted diseases (STDs), etc. These diseases not only affect the quality of life of patients, but also can cause serious complications, such as infertility, ectopic pregnancy, cervical cancer, etc., and infection during pregnancy can also cause premature delivery, premature rupture of membranes and neonatal infection, etc., affecting the health of two generations.

[0003] Genital tract infection usually involves multiple pathogens, including bacteria, viruses, fungi and parasites, etc., and mixed infection and repeated attack are the clinical characteristics of such diseases, and the recurrence rate of mixed infection is much higher than that of single infection, such as the recurrence rate of simple BV and simple VVC after treatment is <10%, while the recurrence rate of BV+VVC mixed infection is more than 40%. The universality of asymptomatic infection is another feature of genital tract infection. Many genital tract infections may have no obvious symptoms in the early stage, but they are still infectious, especially men with disease without obvious symptoms. Research data shows that about 85% of trichomonas vaginalis patients have no symptoms after infection; the clinical symptoms of gonococcal infection in adult women are atypical, and 50% of asymptomatic infections can be found only when complications (such as pelvic inflammatory disease) occur; about 70%~75% of women infected with chlamydia trachomatis have no subjective symptoms and are easily ignored. Therefore, timely and accurate diagnosis, standardized individualized treatment, synchronous treatment of sexual partners, consideration of drug resistance and attention to vaginal ecological balance are needed for genital tract infection.

[0004] The traditional genital tract pathogen detection methods commonly used in the clinic at present include smear microscopy, culture method, serological detection, etc. Smear microscopy is a method of observing cells and microorganisms in a wet film under a microscope for diagnosis, which is simple and rapid, but has relatively low sensitivity and specificity, and requires higher experience of the operator. Culture method can provide diagnostic information, but has limitations such as long time consumption, high requirement for experimental conditions, easy pollution, etc., and is only suitable for a small part of pathogens. Serological detection is simple and rapid in operation, but also has the problem of low sensitivity, which can easily cause missed detection and misdiagnosis.

[0005] Compared with traditional detection methods, nucleic acid detection methods have higher sensitivity and specificity, can significantly improve the detection rate of pathogens, and help clinical implementation of early diagnosis and timely treatment, and gradually become the mainstream method for detection of reproductive tract pathogens. At present, the nucleic acid detection techniques applied to reproductive tract pathogens mainly include fluorescent quantitative PCR, second-generation sequencing, PCR-reverse dot blotting method, etc. The second-generation sequencing technology has the advantages of multiple target detection and good specificity, but has the disadvantages of complicated operation, slow speed, high cost and low detection throughput. The PCR-reverse dot blotting method has the advantages of relatively high detection throughput and low cost, but has the disadvantages of fewer detection targets, complicated operation and slow speed. The fluorescent quantitative PCR technology has the advantages of simple operation, fast speed, low cost, high sensitivity and good specificity, and is widely used in clinics. However, the fluorescent quantitative PCR technology for detection of reproductive tract pathogens currently used in clinics has the disadvantages of fewer detection targets and lack of consideration of vaginal ecological balance, and cannot meet the clinical needs. SUMMARY

[0006] In order to solve at least one of the above problems, the present application provides a primer probe combination, a kit and an application for detecting reproductive tract pathogenic microorganisms.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical means:

[0008] The first aspect of the present application provides a primer probe combination for detecting reproductive tract pathogenic microorganisms, wherein the primer probe combination comprises primer probe sequences as shown in SEQ ID NO. 1-SEQ ID NO. 96, and the reproductive tract pathogens comprise Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma genitalium, Mycoplasma genitalium, Candida albicans, Candida glabrata, Candida tropicalis, Candida auris, Candida krusei, Candida dubliniensis, Candida parapsilosis, Streptococcus group B, Corynebacterium parvum, Bartonella henselae, Actinomyces odontolyticus, Trichomonas vaginalis, Entamoeba, Neisseria gonorrhoeae, Haemophilus ducreyi, Chlamydia trachomatis, Herpes simplex virus type II, Herpes simplex virus type I, Treponema pallidum, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus iners, Bifidobacterium, Atopobium vaginae, Gardnerella vaginalis, Leptothrix, and a human internal standard. The primer probe sequences as shown in SEQ ID NO. 1-SEQ ID NO. 96, the primer probe sequence corresponding to each pathogenic microorganism, and the SEQ ID NO. number from small to large are in turn a forward primer sequence, a probe sequence and a reverse primer sequence.

[0009] In some embodiments of the present application, the primer probe combination is divided into 8 primer probe large groups: the first primer probe large group includes primer probe sequences of Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, as shown in SEQ ID NO. 1-SEQ ID NO. 12, wherein the sequences with serial numbers 2, 5, 8, and 11 are probe sequences; the second primer probe large group includes primer probe sequences of Candida albicans, Candida glabrata, Candida tropicalis, Candida auris, as shown in SEQ ID NO. 13-SEQ ID NO. 24, wherein the sequences with serial numbers 14, 17, 20, and 23 are probe sequences; the third primer probe large group includes primer probe sequences of Candida krusei, Candida dubliniensis, Candida parapsilosis, and human internal standard, as shown in SEQ ID NO. 25-SEQ ID NO. 33, SEQ ID NO. 94-SEQ ID NO. 96, wherein the sequences with serial numbers 26, 29, 32, and 95 are probe sequences; the fourth primer probe large group includes primer probe sequences of Streptococcus group B, Corynebacterium parvum, Bartonella henselae, Actinomyces israelii, as shown in SEQ ID NO. 34-SEQ ID NO. 45, wherein the sequences with serial numbers 35, 38, 41, and 44 are probe sequences; the fifth primer probe large group includes primer probe sequences of Trichomonas vaginalis, Entamoeba, Neisseria gonorrhoeae, Haemophilus ducreyi, as shown in SEQ ID NO. 46-SEQ ID NO. 57, wherein the sequences with serial numbers 47, 50, 53, and 56 are probe sequences; the sixth primer probe large group includes primer probe sequences of Chlamydia trachomatis, Herpes simplex virus type II, Herpes simplex virus type I, Treponema pallidum, as shown in SEQ ID NO. 58-SEQ ID NO. 69, wherein the sequences with serial numbers 59, 62, 65, and 68 are probe sequences; the seventh primer probe large group includes primer probe sequences of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus iners, as shown in SEQ ID NO. 70-SEQ ID NO. 81, wherein the sequences with serial numbers 71, 74, 77, and 80 are probe sequences; the eighth primer probe large group includes primer probe sequences of Bifidobacterium, Atopobium vaginae, Gardnerella vaginalis, Leptothrix, as shown in SEQ ID NO. 82-SEQ ID NO. 93, wherein the sequences with serial numbers 83, 86, 89, and 92 are probe sequences.

[0010] In some embodiments of the present application, the 5' end of the probe sequence of the primer probe combination is provided with a fluorescent group, and the 3' end is provided with a quenching group. In the same primer probe large group, the fluorescent groups on the probes of different pathogenic microorganisms are different.

[0011] In some embodiments of the present application, the fluorescent group is one of FAM, HEX, VIC, ROX and CY5, and the quenching group is one of BHQ1, MGB and BHQ2.

[0012] The second aspect of the present application provides a detection kit for pathogenic microorganisms in the genital tract, comprising the primer probe combination of the first aspect.

[0013] When the primer probe combination of the first aspect is used to produce the detection kit for pathogenic microorganisms in the genital tract, PCR reagents are further included, preferably, the PCR detection reagent is Lyo-Ready qPCR Mix reagent produced by Midean Company.

[0014] The third aspect of the present application provides the use of the kit of the second aspect in the simultaneous detection of pathogenic microorganisms in the genital tract and the evaluation of the ecological environment of the genital tract. In some embodiments of the present application, the pathogenic microorganisms in the genital tract include 27 kinds of pathogens that can cause diseases in the genital tract, such as Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Candida albicans, Candida glabrata, Candida tropicalis, Candida auris, Candida krusei, Candida dubliniensis, Candida parapsilosis, Streptococcus group B, Corynebacterium minutissimum, Bartonella henselae, Actinomyces israelii, Trichomonas vaginalis, Entamoeba, Neisseria gonorrhoeae, Haemophilus ducreyi, Chlamydia trachomatis, Herpes simplex virus type II, Herpes simplex virus type I, Treponema pallidum, Bifidobacterium, Atopobium vaginae, Gardnerella vaginalis, Leptothrix, and the like.

[0015] Among them, (1) 7 kinds of pathogens related to bacterial vaginosis: Gardnerella vaginalis, Atopobium vaginae, Leptothrix, Bifidobacterium, Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis.

[0016] (2) 3 kinds of pathogens related to aerobic vaginitis: Streptococcus group B, Corynebacterium minutissimum, Actinomyces israelii.

[0017] (3) 7 kinds of pathogens related to vulvovaginal candidiasis: Candida albicans, Candida glabrata, Candida tropicalis, Candida auris, Candida krusei, Candida dubliniensis, Candida parapsilosis.

[0018] (4) 7 kinds of pathogens related to sexually transmitted diseases: Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi, Mycoplasma genitalium, Herpes simplex virus type 1, Herpes simplex virus type 2.

[0019] (5) Parasites causing trichomonas vaginitis: Trichomonas vaginalis.

[0020] (6) Other pathogens causing reproductive tract infection: amoeba, bartonella henselae.

[0021] (7) 4 common vaginal lactobacillus: lactobacillus crispatus, lactobacillus gasseri, lactobacillus jensenii, lactobacillus iners. By detecting the 4 main vaginal lactobacillus, the evaluation of vaginal ecological environment can be carried out.

[0022] For the detection of the above 31 pathogenic microorganisms, not only the main pathogens of reproductive tract infection can be covered, but also the 4 common vaginal lactobacillus, so that the evaluation of vaginal ecological environment can be met while the pathogen infection is detected.

[0023] In some embodiments of the present application, the kit is used for simultaneous detection of reproductive tract pathogenic microorganisms and evaluation of vaginal ecological environment, comprising the following steps:

[0024] (1) Dilute the primers and probes in the primer probe combination of the first aspect;

[0025] (2) Prepare the primer probe working solution of the reproductive tract pathogens and human internal standard according to the first to eighth primer probe groups of the first aspect;

[0026] (3) Prepare the PCR reaction system, add the sample and perform fluorescence PCR detection;

[0027] The detection result judgment standard is: the Ct value of the human internal standard is ≤31, otherwise the PCR reaction is invalid and needs to be re-detected; the Ct value corresponding to each target is ≤36, and the amplification curve is a typical S-shaped curve, which is judged as positive for the corresponding pathogenic microorganism.

[0028] In some embodiments of the present application, the sample is a vaginal swab sample or a cervical exfoliative cell sample.

[0029] In some embodiments of the present application, the final concentration of the primer of the reproductive tract pathogen in the PCR reaction system is 1-1.33 μM, and the final concentration of the probe is 0.33-1.33 μM; the final concentration of the primer of the human internal standard in the PCR reaction system is 1.33 μM, and the final concentration of the probe is 0.6 μM.

[0030] In some embodiments of the present application, in the reaction system, the volume ratio of PCR buffer solution to primer probe working solution is 2:1; the PCR reaction program is: 50℃ 5min, 95℃ 2min, 95℃ 3s 54℃ 5s, 5 cycles, 95℃ 3s 58℃ 5s, 40 cycles.

[0031] Advantages of the present application

[0032] Compared with the prior art, the present application has the following advantages:

[0033] The application is based on fluorescent quantitative PCR technology, and can be used for detecting 31 pathogenic microorganisms in a genital tract sample, including 27 pathogenic microorganisms that can cause genital tract diseases and 4 common vaginal lactobacilli. The application not only can cover the main pathogenic microorganisms of genital tract infection, but also can meet the evaluation of the vaginal ecological environment. The joint detection of multiple pathogenic microorganisms can improve the accuracy of diagnosis and promote precision medicine. The application can be widely applied in gynecology, obstetrics, and reproductive medicine department, and can be used for monitoring and diagnosing the infection of the genital tract.

[0034] Compared with the detection time of more than 1 hour of the current detection technology of genital tract pathogenic microorganisms based on fluorescent quantitative PCR, the detection time of the application is shortened to about 40 minutes after screening the optimal primer probe combination and detection reagent, and the application has good specificity and sensitivity. The main performance is that there is no cross between the detected pathogenic microorganisms, and the minimum detection limit of Candida auris, Corynebacterium parvum, Amoeba, Lactobacillus jensenii, and Leptomyxid can reach 200 copies / mL, and the minimum detection limit of other pathogenic microorganisms can reach 100 copies / mL. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The figure shows the amplification curve of the three groups of primer probe combinations designed by the application for each pathogenic microorganism. Red represents primer probe combination 1, yellow represents primer probe combination 2, and blue represents primer probe combination 3.

[0036] Figure 2 The figure shows the amplification efficiency detection result comparison chart of two groups of different PCR detection reagents of the application. Red represents detection reagent 1: Lyo-Ready qPCR Mix reagent produced by Medigen Company, and blue represents detection reagent 2: TaqMan™ Lyo-ready qPCR Master Mix reagent produced by Thermo Fisher Company.

[0037] Figure 3 The figure shows the specificity detection result comparison chart of two groups of different PCR detection reagents of the application. Blue represents the FAM fluorescence channel, green represents the HEX fluorescence channel, orange represents the ROX fluorescence channel, and red represents the CY5 fluorescence channel.

[0038] Figure 4 The figure shows the amplification curve of the minimum detection limit of each pathogenic microorganism and the human internal standard in the application.

[0039] Figure 5 The figure shows the amplification curve of sample 1 in Example 5 of the application.

[0040] Figure 6 The figure shows the amplification curve of sample 2 in Example 5 of the application.

[0041] Figure 7 Figure 5 shows the amplification curve of sample 3 in Example 5 of the present application.

[0042] Figure 8 Figure 6 shows the amplification curve of sample 4 in Example 5 of the present application. DETAILED DESCRIPTION

[0043] The following examples are put forth so as to demonstrate preferred embodiments of the application. Those skilled in the art will recognize that the examples disclosed herein represent the inventive discoveries made by the inventors and are to be considered as preferred methods for practicing the application. However, those skilled in the art will recognize, in light of the teachings herein, that many modifications can be made to the particular embodiments disclosed without departing from the spirit or scope of the application.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the materials referred to in this disclosure are incorporated by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the following claims.

[0045] The technical solutions of the present application will be further described in detail below in conjunction with the specific embodiments.

[0046] Example 1 primer probe design and screening

[0047] The gene sequences of each pathogenic microorganism were searched in the NCBI database, and the conserved regions of each gene sequence were searched by sequence alignment. Primers and probes were designed according to the conserved regions of each gene sequence, and three groups of primer and probe combinations were designed for each pathogenic microorganism. PCR amplification was performed on each pathogenic microorganism and human internal standard using the three groups of designed primer and probe combinations, and the amplification curve results obtained are shown in Figure 1. Figure 1

[0048] The results show that the amplification effect of primer and probe combination 1 is the best, and it is used as the primer and probe combination of the present application.

[0049] The primer and probe sequences of the 31 pathogenic microorganisms and human internal standard screened out are shown in Table 1 below.

[0050] Table 1 primer and probe sequence information

[0051] ​

[0052] Preparation of primer probe working solution

[0053] 1. Dilution of primer probe

[0054] Dilute primer probe dry powder to 20 μM with 1 × TE Buffer, calculation method as follows:

[0055] Dilute according to the number of nmoles: 1 × TE Buffer added per tube = number of nmoles × 50 (μL); vortex for about 30 s after adding 1 × TE Buffer, and centrifuge at low speed for a short time.

[0056] 2. Preparation of primer probe working solution

[0057] Prepare primer probe working solution 1-8 according to Table 2-9, and vortex the prepared solution and label it.

[0058] Table 2 Composition list of primer probe working solution 1

[0059]

[0060] Table 3 Composition list of primer probe working solution 2

[0061]

[0062] Table 4 Composition list of primer probe working solution 3

[0063]

[0064] Table 5 Composition list of primer probe working solution 4

[0065]

[0066] Table 6 Composition list of primer probe working solution 5

[0067]

[0068] Table 7 Composition list of primer probe working solution 6

[0069]

[0070] Table 8 Composition list of primer probe working solution 7

[0071]

[0072] Table 9 Composition list of primer probe working solution 8

[0073]

[0074] Example 3 Screening of PCR detection reagent

[0075] System 7 and system 8 were respectively subjected to amplification efficiency and specificity test by PCR detection reagent 1: Lyo-Ready qPCR Mix reagent produced by Medigen, and PCR detection reagent 2: TaqMan™ Lyo-ready qPCR Master Mix reagent produced by Thermo, and other experimental conditions were the same:

[0076] In the amplification efficiency test, the detection template was the corresponding pathogenic microorganism plasmid in system 7 and system 8, and the amplification efficiency results were as shown in Figure 2 In the specificity test, no positive template was added, and sterile nuclease-free water was used instead, and the detection results were as shown in Figure 3 .

[0077] From Figure 2 and Figure 3 It can be seen from the results that there is no significant difference between the two PCR detection reagents in terms of amplification efficiency, but PCR detection reagent 2 has non-specific amplification.

[0078] The above results show that the detection results of PCR detection reagent 1 are better than those of PCR detection reagent 2, so we choose to use PCR detection reagent 1: Lyo-Ready qPCR Mix reagent produced by Medigen for subsequent experiments.

[0079] Example 4 PCR detection process

[0080] The PCR detection reagent used in the present application is Lyo-Ready qPCR Mix reagent produced by Medigen.

[0081] 1. Nucleic acid extraction (PCR II room)

[0082] According to the instructions of the nucleic acid extraction or purification reagent (magnetic bead method) produced by Jiangsu Mule Biological Technology Co., Ltd. (Record No.: Jiangsu TCM Preparation 20240008), the sample to be tested was extracted to prepare nucleic acid. The sample types suitable for use include female vaginal swab samples and cervical cell shedding samples.

[0083] 2. Configuration of reaction system (PCR I room)

[0084] Reaction systems 1-8 were prepared according to Table 10, and after preparation, they were aliquoted into PCR reaction wells at 15 μL / well and transferred to the PCR II room.

[0085] Table 10 Preparation of reaction system

[0086]

[0087] 3. Add sample (PCR II room)

[0088] The nucleic acid prepared in 4.1 was added to reaction systems 1-8 at 5 μL / well, and after the addition was completed, it was transferred to the PCR III room for machine detection.

[0089] 4. Machine detection (PCR III room)

[0090] The reaction tube was placed in a fluorescent PCR detector, and the PCR reaction program was set according to Table 11 for detection. The collection of fluorescent signals was set as FAM, HEX / VIC, ROX and CY5, the data collection was set at 58°C, and the reaction volume of each well was set to 20 μL.

[0091] Table 11 PCR reaction program

[0092]

[0093] 5. Result analysis

[0094] After the reaction was completed, the instrument automatically saved the results, and after the image was analyzed, the Start value, End value and Threshold value of the Baseline were adjusted (which can be adjusted by oneself, the Start value can be between 3-15, and the End value is between 5-20). The Ct value of the HEX / VIC fluorescence channel (human internal standard) of system 3 should be ≤31, otherwise the PCR reaction is invalid and needs to be re-detected. If the above conditions are met, analyze according to the following method:

[0095] When the Ct value corresponding to each target is ≤Cut-Off value and the amplification curve is a typical S-shaped curve, it is judged as positive for the corresponding pathogenic microorganism; the corresponding relationship between each reaction system fluorescence channel and detection target is shown in Table 12.

[0096] Table 12 Positive judgment value of each pathogenic microorganism and human internal standard

[0097]

[0098] Example 5 Test of detection limit

[0099] The plasmids of each pathogenic microorganism were selected as simulated samples for detection limit test, and the specific scheme was as follows: the known copy number of each pathogenic microorganism plasmid was diluted to 200 copies / mL, 100 copies / mL and 50 copies / mL with negative samples, respectively, each plasmid was extracted and detected 20 times, and the lowest concentration with 95% positive detection rate was taken as the lowest detection limit. The detection results of each pathogenic microorganism are shown in Table 13.

[0100] Table 13 Detection results of the lowest detection limit

[0101]

[0102] The results show that the minimum detection limit of Candida auris, Corynebacterium parvum, amoeba, Lactobacillus jensenii and Leptomyxidium is 200 copies / mL, and the minimum detection limit of other pathogenic microorganisms is 100 copies / mL. The amplification curve of each pathogenic microorganism and the human internal standard is shown in Figure 4 .

[0103] Example 6: Detection of clinical samples

[0104] Four clinical samples, sample 1, sample 2, sample 3 and sample 4, were selected for detection by the related method of the application. The detection Ct values are shown in Table 14, and the amplification curve is shown in Figures 5 to 8 .

[0105] Table 14: Detection results of clinical samples

[0106]

[0107] All the documents mentioned in the present application are incorporated by reference in the present application, as if each document is incorporated by reference individually. In addition, it should be understood that, after reading the above teaching of the present application, those skilled in the art can make various modifications or changes to the present application, and these equivalent forms also fall within the scope defined by the present application.

Claims

1. The application of primer-probe combinations in the preparation of kits for simultaneous detection of reproductive tract pathogens and assessment of the vaginal ecological environment, characterized in that, The primer-probe combination includes primer-probe sequences as shown in SEQ ID NO.1-SEQ ID NO.

96. The primer-probe combination is divided into eight major groups: the first major group includes primer-probe sequences for *Ureaplasma microsporum*, *Ureaplasma urealyticum*, *Mycoplasma hominis*, and *Mycoplasma genitalium*, as shown in SEQ ID NO.1-SEQ ID NO.12, where sequences 2, 5, 8, and 11 are probe sequences; the second major group includes primer-probe sequences for *Candida albicans*, *Candida glabrata*, *Candida tropicalis*, and *Candida auris*, as shown in SEQ ID NO.13-SEQ ID NO.24, where sequences 14, 17, 20, and 23 are probe sequences; the third major group includes primer-probe sequences for *Candida krusei*, *Candida dublina*, *Candida parapsilosis*, and human internal standards, as shown in SEQ ID NO.25-SEQ ID NO.33 and SEQ ID NO.94-SEQ ID NO.

96. As shown in SEQ ID NO. 96, sequences 26, 29, 32, and 95 are probe sequences; the fourth primer-probe group includes primer-probe sequences for Group B Streptococcus, Corynebacterium pulmonale, Bartonella henneri, and Actinomyces ylangis, as shown in SEQ ID NO. 34-SEQ ID NO. 45, where sequences 35, 38, 41, and 44 are probe sequences; the fifth primer-probe group includes primer-probe sequences for Trichomonas vaginalis, Entamoeba histolytica, Neisseria gonorrhoeae, and Haemophilus ducreyi, as shown in SEQ ID NO. 46-SEQ ID NO. 57, where sequences 47, 50, 53, and 56 are probe sequences; the sixth primer-probe group includes primer-probe sequences for Chlamydia trachomatis, Herpes simplex virus type II, Herpes simplex virus type I, and Treponema pallidum, as shown in SEQ ID NO. 58-SEQ ID NO.

59. The sequence shown in NO. 69 includes sequences 59, 62, 65, and 68, which are probe sequences; the seventh primer-probe group includes primer-probe sequences for *Lactobacillus curvatureii*, *Lactobacillus janniae*, *Lactobacillus gasseri*, and *Lactobacillus indolentus*, as shown in SEQ ID NO. 70-SEQ ID NO. 81, where sequences 71, 74, 77, and 80 are probe sequences; the eighth primer-probe group includes primer-probe sequences for *Bifidobacterium*, *Atopobacterium vaginalis*, *Gardnerella vaginalis*, and *Trichophyton*, as shown in SEQ ID NO. 83-SEQ ID NO. 93, where sequences 83, 86, 89, and 92 are probe sequences; The application includes the following steps: (1) Dilute the primers and probes in the primer-probe combination; (2) Prepare primer and probe working solutions for reproductive tract pathogens and human internal standards according to the first to eighth primer and probe groups respectively; (3) Prepare the PCR reaction system, add the sample, and perform fluorescent PCR detection; In the reaction system, the volume ratio of PCR buffer to primer and probe working solution is 2:1; the PCR reaction program is as follows: 50℃ for 5 min, 95℃ for 2 min, 95℃ for 3 s, 54℃ for 5 s, 5 cycles, 95℃ for 3 s, 58℃ for 5 s, 40 cycles. The criteria for judging the test results are as follows: if the Ct value of the human internal standard is ≤31, the PCR reaction is considered invalid and needs to be retested; if the Ct value of each other target is ≤36 and the amplification curve is a typical S-shaped curve, it is judged as positive for the corresponding pathogen.

2. The application according to claim 1, characterized in that: The probe sequence of the primer-probe combination has a fluorescent group at the 5' end and a quenching group at the 3' end. Within the same primer-probe group, the fluorescent groups on the probes of different pathogenic microorganisms are different.

3. The application according to claim 2, characterized in that: The fluorescent group is one of FAM, HEX, VIC, ROX and CY5, and the quenching group is one of BHQ1, MGB and BHQ2.

4. The application according to claim 1, characterized in that, The sample is either a vaginal swab or a cervical exfoliated cell sample.

5. The application according to claim 1, characterized in that: The final concentration of the primers for the reproductive tract pathogens in the PCR reaction system is 1-1.33 μM, and the final concentration of the probe is 0.33-1.33 μM; the final concentration of the primers for the human internal standard in the PCR reaction system is 1.33 μM, and the final concentration of the probe is 0.6 μM.

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