Primer probe combination for detecting genital tract pathogenic microorganisms, kit and application

Abstract

The invention discloses a primer probe combination and a kit for detecting genital tract pathogenic microorganisms and application. The primer probe combination comprises primer probe sequences as shown in SEQ ID NO.1-SEQ ID NO.96, the genital tract pathogens comprise 27 pathogens capable of causing genital tract diseases, such as ureaplasma parvum, ureaplasma urealyticum, mycoplasma hominis, mycoplasma genitalium, candida albicans, candida glabrata, candida tropicalis, candida auricula, candida krusei and candida duberlin, as well as lactobacillus crispatus, lactobacillus jensenii and lactobacillus gasseri; the lactobacillus lactis comprises four common vaginal lactobacillus lactis, and also comprises a human-derived interior label. On the basis of a fluorescent quantitative PCR technology, the kit can be used for simultaneously detecting 27 pathogens capable of causing genital tract diseases and 4 common lactobacillus vaginalis in an in-vitro sample, not only can cover main pathogens of genital tract infection, but also meets the evaluation on the ecological environment of the vagina, the detection time is shortened to about 40 minutes, and meanwhile, the kit has relatively good specificity and sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer-probe combination, a kit and an application thereof for detecting reproductive tract pathogenic microorganisms. Background Art

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

[0003] Reproductive tract infections usually involve multiple pathogens, including bacteria, viruses, fungi, and parasites. Mixed infections and recurrent attacks are the clinical characteristics of this type of disease, and the recurrence rate of mixed infections is much higher than that of single infections. For example, the recurrence rate of simple BV and simple VVC after treatment is <10%, while the recurrence rate of mixed infection of BV+VVC is more than 40%. The prevalence of asymptomatic infection is another characteristic of reproductive tract infection. Many reproductive tract infections may not have obvious symptoms in the early stage, but they are still contagious, especially for men who are not sick. Research data show that about 85% of patients with vaginal trichomoniasis are asymptomatic after infection; the clinical symptoms of gonococcal infection in adult women are atypical, and 50% of asymptomatic infections can only be discovered when complications (such as pelvic inflammatory disease) occur; about 70%~75% of female patients with Chlamydia trachomatis have no conscious symptoms and are easily ignored. Therefore, reproductive tract infections require timely and accurate diagnosis, standardized individualized treatment, synchronous treatment of sexual partners, consideration of drug resistance, and attention to the balance of vaginal ecology.

[0004] At present, the traditional methods commonly used in clinical practice for detecting reproductive tract pathogens include smear microscopy, culture, and serological testing. Smear microscopy is a method of diagnosis by observing cells and microorganisms in wet mounts under a microscope. It is simple and rapid, but its sensitivity and specificity are relatively low, and it requires high experience from the operator. The culture method can provide confirmed information, but it has limitations such as being time-consuming, requiring high experimental conditions, and being susceptible to contamination, and it is only applicable to a small number of pathogens. Serological testing is simple and rapid to operate, but it also has the problem of low sensitivity, which can easily lead to 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, help clinical implementation of early diagnosis and timely treatment, and gradually become the mainstream method for the detection of reproductive tract pathogens. At present, the nucleic acid detection technologies used for reproductive tract pathogens mainly include fluorescent quantitative PCR, second-generation sequencing, PCR-reverse dot hybridization, etc. The second-generation sequencing technology has many detection targets and good specificity, but the operation is cumbersome, the speed is slow, the cost is high, and the detection throughput is low. The detection throughput of PCR-reverse dot hybridization is relatively high and the cost is low, but there are disadvantages such as fewer detection targets, cumbersome operation, and slow speed. Fluorescent quantitative PCR technology has the characteristics of simple operation, fast speed, low cost, high sensitivity, good specificity, etc., and is widely used in clinical practice. However, the number of pathogens detected by the fluorescent quantitative PCR technology for reproductive tract pathogens currently used in clinical practice is small, and there is a lack of consideration for the ecological balance of the vagina, which cannot meet clinical needs. Summary of the invention

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

[0007] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a primer-probe combination for detecting pathogenic microorganisms of the reproductive tract, the primer-probe combination comprising primer-probe sequences as shown in SEQ ID NO.1-SEQ ID NO.96, the reproductive tract pathogens comprising Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Candida albicans, Candida glabrata, Candida tropicalis, Candida auris, Candida krusei, Candida dubliniensis, Candida parapsilosis, Group B Streptococcus, Corynebacterium parvum, Bartonella henselae, Actinomyces ilei, Trichomonas vaginalis, Ameba, 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 vaginalis, Gardnerella vaginalis, and Ciliate bacteria, and also comprising a human internal standard. Among them, as shown in the primer probe sequences of SEQ ID NO.1-SEQ ID NO.96, the primer probe sequences corresponding to each pathogenic microorganism have SEQ ID NO. numbers from small to large, namely, forward primer sequence, probe sequence, and reverse primer sequence.

[0008] In some embodiments of the present invention, the primer probe combination is divided into 8 primer probe groups: the first primer probe group includes primer probe sequences of Ureaplasma microti, Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium, as shown in SEQ ID NO.1-SEQ ID NO.12, wherein the sequences numbered 2, 5, 8, and 11 are probe sequences; the second primer probe group includes primer probe sequences of Candida albicans, Candida glabrata, Candida tropicalis, and Candida auris, as shown in SEQ ID NO.13-SEQ ID NO.24, wherein the sequences numbered 14, 17, 20, and 23 are probe sequences; the third primer probe group includes primer probe sequences of Candida krusei, Candida dubliniensis, Candida parapsilosis, and human internal standards, as shown in SEQ ID NO.25-SEQ ID NO.36, wherein the sequences with sequence numbers 26, 29, 32, and 35 are probe sequences; the fourth primer probe group includes primer probe sequences of group B Streptococcus, Corynebacterium parvum, Bartonella henselae, and Actinomyces ilitis, as shown in SEQ ID NO.37-SEQ ID NO.48, wherein the sequences with sequence numbers 38, 41, 44, and 47 are probe sequences; the fifth primer probe group includes primer probe sequences of Trichomonas vaginalis, Ameba, Neisseria gonorrhoeae, and Haemophilus ducreyi, as shown in SEQ ID NO.49-SEQ ID NO.60, wherein the sequences with sequence numbers 50, 53, 56, and 59 are probe sequences; the sixth primer probe group includes primer probe sequences of Chlamydia trachomatis, herpes simplex virus type II, herpes simplex virus type I, and Treponema pallidum, as shown in SEQ ID NO.61-SEQ ID NO.72, wherein the sequences with sequence numbers 62, 65, 68, and 71 are probe sequences; the seventh primer probe group includes primer probe sequences of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners, as shown in SEQ ID NO.73-SEQ ID NO.84, wherein the sequences with sequence numbers 74, 77, 80, and 83 are probe sequences; the eighth primer probe group includes primer probe sequences of Bifidobacterium, Atopobium vaginalis, Gardnerella vaginalis, and Trichoderma, as shown in SEQ ID NO.85-SEQ IDNO.96, wherein the sequences with sequence numbers 86, 89, 92, and 95 are probe sequences.

[0009] In some embodiments of the present invention, 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 group, the fluorescent groups on the probes of different pathogenic microorganisms are different.

[0010] In some embodiments of the present invention, the fluorescent group is one of FAM, HEX, VIC, ROX and CY5, and the quenching group is one of BHQ1, MGB and BHQ2.

[0011] The second aspect of the present invention provides a detection kit for reproductive tract pathogenic microorganisms, comprising the primer-probe combination described in the first aspect.

[0012] When the primer-probe combination described in the first aspect is used to produce a detection kit for reproductive tract pathogenic microorganisms, a PCR reagent is also included. Preferably, the PCR detection reagent is the Lyo-Ready qPCR Mix reagent produced by Median.

[0013] The third aspect of the present invention provides the use of the kit described in the second aspect in the simultaneous detection of genital tract pathogenic microorganisms and the assessment of the ecological environment of the genital tract. In some embodiments of the present invention, the genital tract pathogenic microorganisms include ureaplasma microti, ureaplasma urealyticum, mycoplasma hominis, mycoplasma genitalium, Candida albicans, Candida glabrata, Candida tropicalis, Candida auris, Candida krusei, Candida dubliniensis, Candida parapsilosis, group B streptococcus, Corynebacterium parvum, Bartonella henselae, Actinomyces ilei, Trichomonas vaginalis, amoeba, Neisseria gonorrhoeae, Haemophilus ducreyi, Chlamydia trachomatis, herpes simplex virus type II, herpes simplex virus type I, Treponema pallidum, Bifidobacterium, Atopobium vaginalis, Gardnerella vaginalis, cilia, etc. 27 pathogens that can cause genital tract diseases, as well as four common vaginal lactobacilli, including Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners.

[0014] Among them, (1) 7 pathogens associated with bacterial vaginosis: Gardnerella vaginalis, Atopobium vaginalis, Trichoderma, Bifidobacterium, Ureaplasma parvum, Ureaplasma urealyticum, and Mycoplasma hominis.

[0015] (2) Three pathogens associated with aerobic vaginitis: Group B Streptococcus, Corynebacterium parvum, and Actinomyces erythrocytes.

[0016] (3) Seven pathogens associated with vulvovaginal candidiasis: Candida albicans, Candida glabrata, Candida tropicalis, Candida auris, Candida krusei, Candida dubliniensis, and Candida parapsilosis.

[0017] (4) Seven pathogens associated with sexually transmitted diseases: Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi, Mycoplasma genitalium, herpes simplex virus type 1, and herpes simplex virus type 2.

[0018] (5) Parasites that cause trichomoniasis: Trichomonas vaginalis.

[0019] (6) Other pathogens that cause reproductive tract infections: Ameba and Bartonella henselae.

[0020] (7) Four common vaginal lactobacilli: Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus iners. The vaginal ecological environment can be evaluated by detecting the four main vaginal lactobacilli.

[0021] The detection of the above 31 pathogenic microorganisms not only covers the main pathogens of reproductive tract infections, but also includes 4 common vaginal lactobacilli, which can meet the assessment of the vaginal ecological environment while detecting pathogen infections.

[0022] In some embodiments of the present invention, the kit is used to simultaneously detect reproductive tract pathogenic microorganisms and vaginal ecological environment assessment, comprising the following steps: (1) diluting the primer probe in the primer probe combination described in the first aspect; (2) preparing primer and probe working solutions for reproductive tract pathogens and human internal standards respectively according to the first to eighth primer and probe groups described in the first aspect; (3) Prepare the PCR reaction system and perform fluorescence PCR detection after adding samples; The test result judgment criteria are: the Ct value of the human internal standard is ≤31, otherwise the PCR reaction is considered invalid and needs to be re-tested; when the Ct value corresponding to each other target is ≤36 and the amplification curve is a typical S-shaped curve, it is judged to be positive for the corresponding pathogenic microorganism.

[0023] In some embodiments of the present invention, the sample is a vaginal swab sample or a cervical exfoliated cell sample.

[0024] In some embodiments of the present invention, the final concentration of the primers for 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.

[0025] In some embodiments of the present invention, in the reaction system, the volume ratio of PCR buffer to primer probe working solution is 2:1; the PCR reaction program is: 50°C 5min, 95°C 2min, 95°C 3s 54°C 5s, 5 cycles, 95°C 3s 58°C 5s, 40 cycles.

[0026] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: The present invention is based on fluorescent quantitative PCR technology and can be used to detect 31 pathogenic microorganisms in reproductive tract samples in vitro, including 27 pathogens that can cause reproductive tract diseases and 4 common vaginal lactobacilli. It can not only cover the main pathogens of reproductive tract infections, but also meet the assessment of the vaginal ecological environment; multiple pathogens can be detected jointly to improve the accuracy of diagnosis and promote precision medicine; it can be widely used in multiple departments such as gynecology, obstetrics, and reproduction to monitor and diagnose reproductive tract infections.

[0027] Compared with the current reproductive tract pathogen detection technology based on fluorescent quantitative PCR, which has a detection time of more than 1 hour, the present invention shortens the detection time to about 40 minutes after screening the optimal primer probe combination and detection reagent, and has good specificity and sensitivity, which is mainly manifested in that there is no crossover between the detected pathogenic microorganisms, and the minimum detection limit of Candida auris, Corynebacterium parvum, Amoeba, Lactobacillus jensenii, and Ciliate can reach 200 copies / mL, and the minimum detection limit of other pathogenic microorganisms can reach 100 copies / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure shows the screening amplification curves of three sets of primer-probe combinations designed for various pathogenic microorganisms in the present invention; wherein the red one is primer-probe combination 1, the yellow one is primer-probe combination 2, and the blue one is primer-probe combination 3; Figure 2 The figure shows the comparison of the amplification efficiency test results of two groups of different PCR detection reagents of the present invention; wherein the red one is detection reagent 1: Lyo-Ready qPCR Mix reagent produced by Maidian, and the blue one is detection reagent 2: TaqMan™ Lyo-ready qPCR Master Mix reagent produced by Thermo Fisher; Figure 3 A comparison chart of the specificity detection results of two groups of different PCR detection reagents of the present invention is shown; wherein the blue is the FAM fluorescence channel; the green is the HEX fluorescence channel; the orange is the ROX fluorescence channel; and the red is the CY5 fluorescence channel; Figure 4 The minimum detection limit of each pathogenic microorganism in the present invention and the amplification curve of the human internal standard are shown; Figure 5 The amplification curve diagram of sample 1 in Example 5 of the present invention is shown; Figure 6 The amplification curve diagram of sample 2 in Example 5 of the present invention is shown; Figure 7 The amplification curve diagram of sample 3 in Example 5 of the present invention is shown; Figure 8 The amplification curve diagram of sample 4 in Example 5 of the present invention is shown. DETAILED DESCRIPTION

[0029] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0030] 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 the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0031] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0032] Example 1 Primer probe design and screening The gene sequences of each pathogenic microorganism were searched in the NCBI database, and the conserved regions of each gene sequence were searched through sequence comparison. Primers and probes were designed for the conserved regions of each gene sequence. Three sets of primer-probe combinations were designed for each pathogenic microorganism. The three designed primer-probe combinations were used to perform PCR amplification on each pathogenic microorganism and the human internal standard. The amplification curve results were as follows: Figure 1 shown.

[0033] The results showed that primer-probe combination 1 had the best amplification effect and was used as the primer-probe combination of the present application.

[0034] The specific information of the primer and probe sequences of the 31 pathogenic microorganisms and human internal standards that were screened are shown in Table 1 below.

[0035] Table 1 Primer probe sequence information

[0036] Example 2 Configuration of primer probe working solution 1. Dilution of primers and probes Primer probe powder is diluted to 20 μM with 1×TE Buffer. The calculation method is as follows: Dilute according to the number of nmoles: 1×TE Buffer added to each tube = number of nmoles × 50 (μL); after adding 1×TE Buffer, vortex for about 30 seconds to evenly mix, and briefly centrifuge at low speed.

[0037] 2. Preparation of primer and probe working solution Primer probe working solutions 1 to 8 are prepared according to Table 2-9. The prepared solutions are vortexed to mix well and labeled.

[0038] Table 2 Composition list of primer probe working solution 1

[0039] Table 3 Composition list of primer probe working solution 2

[0040] Table 4 Composition list of primer probe working solution 3

[0041] Table 5 Composition list of primer probe working solution 4

[0042] Table 6 Composition list of primer probe working solution 5

[0043] Table 7 Composition list of primer probe working solution 6

[0044] Table 8 Composition list of primer probe working solution 7

[0045] Table 9 Composition list of primer probe working solution 8

[0046] Example 3 Screening of PCR detection reagents System 7 and system 8 were selected to test the amplification efficiency and specificity using PCR detection reagent 1: Lyo-Ready qPCR Mix produced by Meridian and PCR detection reagent 2: TaqMan™ Lyo-ready qPCR Master Mix produced by Thermo Fisher Scientific. Other experimental conditions were the same: In the amplification efficiency test, the detection template is the corresponding pathogenic microorganism plasmid in system 7 and system 8. The amplification efficiency results are as follows: Figure 2 In the specificity test, no positive template was added, and sterile nuclease-free water was used instead. The test results are shown in Figure 3 shown.

[0047] Depend on Figure 2 and Figure 3 The results show that there is no significant difference in amplification efficiency between the two PCR detection reagents, but PCR detection reagent 2 has non-specific amplification.

[0048] The above results show that the detection results of PCR detection reagent 1 are better than those of PCR detection reagent 2, so we chose to use PCR detection reagent 1: Lyo-Ready qPCR Mix reagent produced by Median for subsequent experiments.

[0049] Example 4 PCR detection process The PCR detection reagent used in the present invention is the Lyo-Ready qPCR Mix reagent produced by Median.

[0050] 1. Nucleic acid extraction (PCR II room) According to the instructions of the nucleic acid extraction or purification reagent (magnetic bead method) (registration number: Sutai Xiebei 20240008) produced by Jiangsu Mole Biotechnology Co., Ltd., the samples to be tested were extracted and nucleic acids were prepared. Applicable sample types include female vaginal swab samples and cervical exfoliated cell samples.

[0051] 2. Configuration of reaction system (PCRⅠ room) Reaction systems 1 to 8 were prepared according to Table 10. After preparation, 15 μL / well was dispensed into PCR reaction wells and transferred to PCR II chamber.

[0052] Table 10 Preparation of reaction system

[0053] 3. Sample addition (PCR II room) Add the nucleic acid prepared in 4.1 to reaction systems 1-8 at 5 μL / well. After the addition is completed, transfer to the PCR III chamber for on-machine detection.

[0054] 4On-machine testing (PCR III room) Place the reaction tube in a fluorescent PCR detector and set the PCR reaction program for detection according to Table 11. The collection of fluorescent signals is set to FAM, HEX / VIC, ROX and CY5, the data collection is set at 58°C, and the reaction volume of each well is set to 20 μL.

[0055] Table 11 PCR reaction program

[0056] 5 Results Analysis After the reaction is completed, the instrument automatically saves the results. After analyzing the image, adjust the Start value, End value and Threshold value of the Baseline (you can adjust it yourself, 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 considered invalid and needs to be retested. If the above conditions are met, analyze according to the following method: When the Ct value corresponding to each target is ≤ the Cut-Off value and the amplification curve is a typical S-shaped curve, the corresponding pathogenic microorganism is judged to be positive; the corresponding relationship between the fluorescence channel of each reaction system and the detection target is shown in Table 12.

[0057] Table 12 Positive judgment values ​​of pathogenic microorganisms and human internal standards

[0058] Example 5 Testing of detection limit The plasmids of each pathogenic microorganism were selected as simulated samples for the detection limit test. The specific scheme is as follows: the plasmids of each pathogenic microorganism with known copy number were diluted to 200 copies / mL, 100 copies / mL and 50 copies / mL respectively with negative samples, and each plasmid was extracted and tested 20 times repeatedly, and the lowest concentration with a positive detection rate of 95% was taken as the lowest detection limit. The detection results of each pathogenic microorganism are shown in Table 13.

[0059] Table 13 Minimum detection limit test results

[0060] The results showed that the minimum detection limit of Candida auris, Corynebacterium parvum, Amoeba, Lactobacillus jensenii, and Leptotrichia was 200 copies / mL, and the minimum detection limit of other pathogenic microorganisms was 100 copies / mL. The amplification curves of the minimum detection limit of each pathogenic microorganism and the human internal standard are shown in Figure 2. Figure 4 shown.

[0061] Example 6 Clinical sample detection Four clinical samples were selected: Sample 1, Sample 2, Sample 3, and Sample 4 were tested using the methods of the present invention. The Ct values ​​are shown in Table 14, and the amplification curves are shown in Figures 5 to 8 shown.

[0062] Table 14 Clinical sample test results

[0063] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A primer-probe combination for detecting reproductive tract pathogenic microorganisms, characterized in that: The primer-probe combination includes primer-probe sequences as shown in SEQ ID NO.1-SEQ ID NO.96, and the reproductive tract pathogens include Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Candida albicans, Candida glabrata, Candida tropicalis, Candida auris, Candida krusei, Candida dubliniensis, Candida parapsilosis, Group B Streptococcus, Corynebacterium parvum, Bartonella henselae, Actinomyces ilei, Trichomonas vaginalis, Ameba, 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 vaginalis, Gardnerella vaginalis, and Ciliate bacteria, and also includes a human internal standard.

2. The primer-probe combination for detecting reproductive tract pathogenic microorganisms according to claim 1, characterized in that: The primer probe combination is divided into 8 primer probe groups: the first primer probe group includes primer probe sequences of Ureaplasma microti, Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium, as shown in SEQ ID NO.1-SEQ ID NO.12, wherein the sequences with sequence numbers 2, 5, 8, and 11 are probe sequences; the second primer probe group includes primer probe sequences of Candida albicans, Candida glabrata, Candida tropicalis, and Candida auris, as shown in SEQ ID NO.13-SEQ ID NO.24, wherein the sequences with sequence numbers 14, 17, 20, and 23 are probe sequences; the third primer probe group includes primer probe sequences of Candida krusei, Candida dubliniensis, Candida parapsilosis, and human internal standards, as shown in SEQ ID NO.25-SEQ ID NO.36, wherein the sequences with sequence numbers 26, 29, 32, and 35 are probe sequences; the fourth primer probe group includes primer probe sequences of group B Streptococcus, Corynebacterium parvum, Bartonella henselae, and Actinomyces ildoni, as shown in SEQ ID NO.37-SEQ ID NO.48, wherein the sequences with sequence numbers 38, 41, 44, and 47 are probe sequences; the fifth primer probe group includes primer probe sequences of Trichomonas vaginalis, Ameba, Neisseria gonorrhoeae, and Haemophilus ducreyi, as shown in SEQ ID NO.49-SEQ ID NO.60, wherein the sequences with sequence numbers 50, 53, 56, and 59 are probe sequences; the sixth primer probe group includes primer probe sequences of Chlamydia trachomatis, herpes simplex virus type II, herpes simplex virus type I, and Treponema pallidum, as shown in SEQ ID NO.61-SEQ ID NO.72, wherein the sequences with sequence numbers 62, 65, 68, and 71 are probe sequences; the seventh primer probe group includes primer probe sequences of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners, as shown in SEQ ID NO.73-SEQ ID NO.84, wherein the sequences with sequence numbers 74, 77, 80, and 83 are probe sequences; the eighth primer probe group includes primer probe sequences of Bifidobacterium, Atopobium vaginalis, Gardnerella vaginalis, and Trichophyton, as shown in SEQ ID NO.85-SEQ ID NO.96, wherein the sequences with sequence numbers 86, 89, 92, and 95 are probe sequences.

3. A primer-probe combination for detecting reproductive tract pathogenic microorganisms according to claim 2, characterized in that: 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 combination, the fluorescent groups on the probes of different pathogenic microorganisms are different.

4. The primer-probe combination for detecting reproductive tract pathogenic microorganisms 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.

5. A detection kit for reproductive tract pathogenic microorganisms, characterized in that: The invention comprises the primer-probe combination according to any one of claims 1 to 4.

6. Use of the kit according to claim 5 in the simultaneous detection of pathogenic microorganisms in the reproductive tract and the evaluation of the vaginal ecological environment.

7. The use according to claim 6, characterized in that: The steps include: (1) diluting the primer probe in the primer probe combination of claim 1; (2) preparing primer and probe working solutions for reproductive tract pathogens and human internal standards according to the first primer and probe group to the eighth primer and probe group according to claim 2; (3) Prepare the PCR reaction system and perform fluorescence PCR detection after adding samples; The test result judgment criteria are: the Ct value of the human internal standard is ≤31, otherwise the PCR reaction is considered invalid and needs to be re-tested; when the Ct value corresponding to each other target is ≤36 and the amplification curve is a typical S-shaped curve, it is judged to be positive for the corresponding pathogenic microorganism.

8. The use according to claim 7, characterized in that: The sample is a vaginal swab sample or a cervical exfoliated cell sample.

9. The use according to claim 7, characterized in that: The final concentration of the primers for 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.

10. The use according to claim 7, characterized in that: 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°C 5min, 95°C 2min, 95°C 3s 54°C 5s, 5 cycles, 95°C 3s 58°C 5s, 40 cycles.

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