Primer probe combination and kit for rapidly, synchronously and quantitatively detecting genital tract microorganisms and micro-ecological evaluation method thereof

Abstract

According to the primer probe combination, the kit and the micro-ecological evaluation method for rapidly, synchronously and quantitatively detecting the microorganisms in the genital tract, the primer probe combination is designed, and amplification reaction conditions are improved, so that synchronous and quantitative detection of nearly 50 different types and different types of microorganisms in the genital tract can be realized; according to the method, vaginal lactobacillus groups, pathogenic genital tract microorganisms, sexually transmitted microorganisms and cervix uteri microorganisms are covered, 32 kinds of microorganisms can be distinguished, main microorganisms in the genital tract are comprehensively covered, meanwhile, a corresponding micro-ecological evaluation method is designed in a matched mode, various vaginitis, sexually transmitted diseases and HPV are rapidly diagnosed, and the method has the advantages of being simple in operation, convenient to operate and high in practicability. And a basis is provided for clinical treatment and prognosis of genital tract infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a primer-probe combination, a kit and a microecological evaluation method thereof for rapid synchronous quantitative detection of reproductive tract microorganisms. Background Art

[0002] Reproductive tract infection is a common gynecological disease in women and a global social and public health problem. Common reproductive tract infections mainly include vaginitis (bacterial, trichomonal, candidiasis, aerobic vaginitis), sexually transmitted diseases, cervical diseases, etc. The main cause of reproductive tract infection is the invasion of the reproductive tract by various microorganisms such as bacteria, viruses, mycoplasma, fungi, and trichomonas. In the normal vaginal flora, the reduction or absence of Lactobacillus (Lactobacillus crispatus, Lactobacillus agar, Lactobacillus James, and Lactobacillus iners), which are the dominant bacteria, weakens the inhibitory effect on other microorganisms, causing the pathogenic microorganisms to multiply in large numbers, thus leading to vaginitis. Bacterial vaginitis, aerobic vaginitis, Trichomonas vaginitis and candidiasis vaginitis account for more than 70% to 90% of vaginitis cases, among which bacterial vaginitis accounts for about 36% to 60% of patients with vaginitis, aerobic vaginitis accounts for about 9.4% to 23.7% of patients with vaginitis, Trichomonas vaginitis accounts for about 0.3% to 20% of patients with vaginitis, and candidiasis accounts for about 15% to 40% of patients with vaginitis.

[0003] The main microorganisms that cause bacterial vaginitis include Gardnerella vaginalis, Atopobium vaginalis, Prevotella, Mobiluncus, Bacteroides, Giant coccus, bacterial vaginitis-associated bacteria 2 (BVAB2), Ureaplasma urealyticum, Mycoplasma hominis, etc.; the microorganisms that cause aerobic vaginitis mainly include Group A Streptococcus, Group B Streptococcus, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Streptococcus anginosus, and Klebsiella pneumoniae; the microorganism that causes Trichomonas vaginitis is Trichomonas vaginalis; vulvovaginal candidiasis, also known as vulvovaginal candidiasis, is a reproductive tract disease caused by Candida infection, and its pathogenic bacteria are mainly Candida albicans, and other non-albicans such as Candida glabrata, Candida tropicalis, and Candida krusei account for a minority. Sexually transmitted diseases are a class of diseases that are widely prevalent. Gonorrhea is the most common type of disease, and the main microorganism is Neisseria gonorrhoeae. In non-gonococcal urethritis, the common microorganism is Chlamydia trachomatis, and Mycoplasma genitalium can also cause non-gonococcal urethritis. Treponema pallidum is the microorganism that causes syphilis, and more than 95% of syphilis infections are caused by sexual contact. Herpes simplex virus types 1 and 2 can cause vesicular infections in the genitals and perianal areas, causing sexually transmitted diseases. There are more than 200 subtypes of human papillomavirus (HPV) that can infect humans, of which more than 40 can cause human diseases, mainly through contact infection. After persistent infection with HPV, it can cause proliferative lesions of the human reproductive tract mucosa and skin, and clinically manifest as different types of epithelial warts, cervical cancer, anal cancer, vaginal cancer and other malignant tumors. According to the degree of harm caused by HPV, WHO divides HPV into 14 high-risk types: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, 4 medium-high-risk types: HPV 26, 53, 73 and 82, and the rest are low-risk types. Persistent infection with high-risk HPV is the main cause of cervical cancer and its precancerous lesions.

[0004] The above microorganisms may cause single infection or mixed infection with multiple microorganisms. Mixed infection of genital tract microorganisms is often accompanied by the existence of a complex genital tract environment. Compared with single microbial infection, the diagnosis and treatment of mixed infection is more difficult.

[0005] Clinically, vaginal microecological testing mainly includes morphological and functional testing. Traditional morphological and functional testing, culture methods, and serological testing methods are low-cost and easy to operate, but they mainly rely on morphological observation and judgment under a microscope, which not only requires a lot of manpower, but also requires high ability and experience of the inspectors. It is impossible to specifically identify the types and quantification of microorganisms, and it is impossible to comprehensively and quickly diagnose reproductive tract infections caused by multiple microorganisms. Given that it takes a long time to detect mixed infections of reproductive tract microorganisms, the clinic needs a method that can quickly and accurately detect the main microorganisms that cause reproductive tract infections, and diagnose the status of reproductive tract microecology through the test results.

[0006] Real-time fluorescence quantitative PCR technology has advantages such as good sensitivity and specificity, and is currently a commonly used microbial detection method in clinical practice. However, the reagent detection based on molecular diagnostic technology for detecting genital tract infection microorganisms on the market is time-consuming, and most of them are qualitative detection of a single microorganism or several microorganisms that cause a single vaginitis, and cannot cover the main microorganisms of multiple genital tract infections at the same time, and there is no product with rapid quantitative detection and microecological evaluation functions. For example, the primer probe combination provided by CN117925870A can simultaneously detect 11 vaginal microorganisms, including vaginal Gardnerella, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Group B Streptococcus, Trichomonas vaginalis, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus crispatus, and is detected by a PCR detection device with five fluorescence channels, but because only 11 microorganisms can be detected at the same time, it is difficult to comprehensively evaluate the genital tract microecological status.

[0007] Therefore, there is an urgent need to develop a method that can simultaneously and quantitatively detect almost all microorganisms in the reproductive tract within 30 minutes, so as to assist in the diagnosis of various types of reproductive tract infections and evaluate the reproductive tract microecology, which is of great significance for the realization of multiple joint detection of reproductive tract microorganisms and research on sexually transmitted diseases. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a primer probe combination, a kit and a microecological evaluation method for rapid and synchronous quantitative detection of genital tract microorganisms. Specifically, it is based on a multiplex fluorescence PCR method to perform synchronous quantitative detection of nearly 50 different types and types of microorganisms in the genital tract, covering vaginal lactobacillus groups, pathogenic genital tract microorganisms, sexually transmitted microorganisms and cervical microorganisms, and can distinguish 32 of these microorganisms, achieving comprehensive coverage of the main microorganisms in the genital tract. At the same time, a corresponding microecological evaluation method is also designed to quickly diagnose a variety of vaginitis, sexually transmitted diseases and HPV, providing a basis for the clinical treatment and prognosis of genital tract infections.

[0009] On the one hand, the present invention provides a quantitative evaluation system for rapid and synchronous detection of reproductive tract microecology, which is used to analyze the quantitative detection results of microorganisms in samples and evaluate the reproductive tract microecology, thereby realizing auxiliary diagnosis of various reproductive tract infections.

[0010] Furthermore, the system performs quantitative detection of reproductive tract microorganisms based on a standard curve established after multiple PCR amplification using a variety of primer-probe combinations, and is corrected by a high-level quantitative traceability system, so that all target microorganisms can be accurately quantified.

[0011] Furthermore, the system quantitatively detects and analyzes nearly 50 different types and types of reproductive tract microorganisms, and uses a matching evaluation method to evaluate and analyze the reproductive tract microecology, including the determination of the status of the reproductive tract flora and the rapid diagnosis of reproductive tract diseases, including bacterial vaginitis, aerobic vaginitis, vulvovaginal candidiasis, vaginal trichomoniasis, sexually transmitted diseases and HPV.

[0012] Furthermore, the evaluation method is a set of microecological evaluation methods that are most suitable for the present invention, developed by the present invention after quantitative analysis of 50 different types and types of microorganisms and a large amount of data analysis. This method does not need to rely on microscopes and the experience of inspectors to analyze reproductive tract diseases like traditional technologies. It only uses the fixed value proportion of lactobacillus groups and vaginitis-related microorganisms in the sample to achieve efficient, convenient and accurate microecological evaluation, providing a new direction for reproductive tract microecological diagnosis.

[0013] The microecological quantitative evaluation system provided by the present invention realizes the rapid diagnosis of bacterial vaginitis and aerobic vaginitis by calculating the fixed value proportion of lactobacillus group and vaginitis-related microorganisms in the sample. At the same time, the system performs quantitative detection and result interpretation on a variety of microorganisms such as Candida, Trichomonas vaginalis, sexually transmitted disease-related microorganisms, and human papillomavirus in the sample, thereby realizing the rapid diagnosis of external vaginal candidiasis, vaginal trichomoniasis, sexually transmitted diseases, and HPV infection. The present invention uses standard products to compare the quantitative evaluation system of the genital tract microecology with the digital PCR method, and determines the quantitative accuracy of the quantitative evaluation system of the genital tract microecology. The present invention uses clinical samples to compare the quantitative evaluation system of the genital tract microecology with the gold standard method and the tNGS method, and determines the application accuracy of the quantitative evaluation system of the genital tract microecology.

[0014] In another aspect, the present invention provides a primer-probe combination for rapid and synchronous detection of reproductive tract microorganisms, wherein the primer-probe combination comprises any one or more of the following:

[0015] Table 1. Primer-probe combinations for 32 reproductive tract microorganisms

[0016]

[0017] The present invention adopts the method of synchronously monitoring the dominant vaginal flora and the main microorganisms causing genital tract infections to assist in the diagnosis of the patient's genital tract microecological status and provide a basis for the clinical treatment and prognosis of genital tract infections. It covers:

[0018] 1. Four dominant species that are more common in normal vaginal flora (Lactobacillus crispatus, Lactobacillus caninus, Lactobacillus jenne, and Lactobacillus iners);

[0019] 2. 9 bacterial vaginosis microorganisms (Gardnerella vaginalis, Prevotella, Mobiluncus, Bacteroides, Atopobium vaginalis, Giant coccus, Bacterial vaginosis-associated bacteria 2 (BVAB2), Ureaplasma urealyticum, Mycoplasma hominis), which can cover more than 85% of clinical cases of bacterial vaginosis, among which Gardnerella vaginalis, Atopobium vaginalis, and Prevotella are the species with the highest clinical incidence;

[0020] 3. 7 types of aerobic vaginitis microorganisms (Group A Streptococcus, Group B Streptococcus, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Streptococcus anginosus, Klebsiella pneumoniae), which can cover more than 90% of clinical cases of aerobic vaginitis, among which Group B Streptococcus, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis are the species with the highest clinical incidence;

[0021] 4. Testing for vaginal Trichomonas can diagnose whether Trichomonas vaginitis infection occurs;

[0022] 5. Four types of candidal vaginitis microorganisms (Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei, of which only Candida albicans can be typed) cover more than 90% of clinical candidal vaginitis cases, of which 65%-70% are Candida albicans, and 10%-20% are Candida glabrata, Candida tropicalis, and Candida krusei;

[0023] 6. Six sexually transmitted pathogenic microorganisms (Treponema pallidum, Neisseria gonorrhoeae, Chlamydia trachomatis, herpes simplex virus type 1, herpes simplex virus type 2, Mycoplasma genitalium) and 18 human papillomaviruses that cause cervical cancer or genital tract lesions (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 53, 82 and 26, of which only HPV16 and HPV18 can be specifically classified), can cover most of the common sexually transmitted disease microorganisms.

[0024] The present invention designs specific amplification primers and Taqman probes according to the internal standard fragment of the human genome and the conserved regions of the target genes of 32 reproductive tract microorganisms, and determines whether the corresponding microorganisms are detected by the positive and negative nature of the target genes of the reproductive tract microorganisms.

[0025] At present, the number of fluorescence channels of fluorescence quantitative PCR equipment on the market is usually 4 to 6, so multiplex fluorescence quantitative PCR can detect 3-5 pathogens and 1 internal standard gene in one system. Therefore, we grouped the 32 microorganisms for detection, selected 3 to 5 microorganisms in each group for combination, and divided them into different sample tubes for synchronous amplification and quantitative detection. According to the principle of multiplex primer probe design, the primer pairs and probes of 3-5 microorganisms in the same group are labeled with different fluorescent groups, and different channels are selected for detection without interfering with each other.

[0026] Furthermore, the primer-probe combination also includes an internal standard primer-probe combination.

[0027] In some embodiments, the internal standard is the RNase P gene, the upstream primer sequence is shown in SEQ ID NO.127, the downstream primer sequence is shown in SEQ ID NO.128, and the probe sequence is shown in SEQ ID NO.129.

[0028] The present invention can monitor the sample collection, storage and transportation, nucleic acid extraction and PCR amplification process by adding a human genome internal standard, thereby avoiding false negative results.

[0029] Furthermore, the primer-probe combination includes eight groups of primer-probe combinations as shown in the following table, each group can achieve synchronous detection of reproductive tract microorganisms within the group:

[0030] Table 2 Grouping of eight primer-probe combinations

[0031]

[0032] The 32 groups of primer-probe combinations for microorganisms provided by the present invention can also be pre-packaged for use, and the preferred grouping method is to divide them into eight groups as shown in Table 2.

[0033] Furthermore, the sixth group also includes any one or more of the 15 HPV other high-risk typing primer-probe combinations shown in Table 3:

[0034] Table 3. Primer-probe combinations for other high-risk HPV typing

[0035]

[0036] Other high-risk HPV types can also be directly added to the sixth group for simultaneous testing, which can still ensure the accuracy of the test results. Some primer probes of different HPV types can be shared, such as HPV31 and HPV35 probes, HPV56 and 66, HPV39 and 68, HPV33 and 58, HPV51 and 82 primer probes.

[0037] In some methods, 15 other high-risk HPV types and HPV31 in the sixth group are in the same channel and detected by the same fluorescent group. Once one of them is positive, it can be detected and judged as positive.

[0038] In some embodiments, the primer-probe compositions of the first to eighth groups all contain an internal standard, which is the RNase P gene, the upstream primer sequence is shown in SEQ ID NO.127, the downstream primer sequence is shown in SEQ ID NO.128, and the probe sequence is shown in SEQ ID NO.129.

[0039] In some embodiments, the 5' ends of the primer-probe combination and the internal standard probe are respectively labeled with different fluorescent groups, and the 3' ends are labeled with a quenching group.

[0040] The fluorescent group labeled at the 5' end of the specific probe is selected from any one or more of FAM, HEX, VIC, JOE, TET, TAMRA, ROX, QUASAR 705, CY3.5 or CY5; the quenching group is selected from any one or more of BHQ1, BHQ2, BHQ3, TAMRA, DABCYL and MGB.

[0041] In some embodiments, the 5' end of the probe for the microorganism to be detected is labeled with a fluorescent group FAM / VIC / ROX / CY5, and the 5' end of the probe for the internal standard is labeled with a fluorescent group QUASAR 705.

[0042] In another aspect, the present invention provides a kit for rapid and synchronous detection of reproductive tract microorganisms, the kit comprising the primer-probe combination as described above.

[0043] Furthermore, the kit also includes an amplification reaction solution, which includes Taq DNA polymerase and UDG enzyme.

[0044] The present invention introduces a UDG enzyme anti-pollution system into the amplification system, which can effectively avoid the occurrence of false positives.

[0045] Furthermore, the amplification reaction solution also includes PCR buffer, MgCl2, dNTPs and RNase-free water.

[0046] The present invention uses a specific Taq DNA polymerase modified by directed mutagenesis (Thermo Scientific™ DreamTaq™ DNA polymerase is an enhanced version of Taq DNA polymerase), which helps to improve the accuracy and sensitivity of the detection. The reason may be that the specific Taq DNA polymerase modified by directed mutagenesis has a stricter matching start than the conventional Taq enzyme; at the same time, the purity and concentration of the enzyme antibody are improved, which can effectively block the activity of the Taq enzyme; the enzyme antibody also uses a monoclonal antibody in the active center region, and the antibody binding is more stable, which can effectively prevent non-specific amplification and prevent mutual interference between different primer probes.

[0047] In some embodiments, the content of the DNA polymerase and UDG enzyme is 3-10 U / Test.

[0048] In some embodiments, the PCR buffer is 2× Buffer.

[0049] In some embodiments, the concentration of MgCl2 is 0-10 mM; the concentration of dNTPs is 0.5-1 mM, including dATP: dTTP: dCTP: dGTP: dUTP=1:1:1:1:1.

[0050] The RNase-free water is used to make up the volume of the reaction solution, and varies according to the concentration and amount of MgCl2, dNTPs, etc.

[0051] In some embodiments, the kit also includes standard substances for quantitative detection, and the quantitative standard substances are WHO standards, national quantitative standards, third-party quantitative standards, or nucleic acids of target microbial samples, pseudovirus nucleic acids, or plasmid DNA with determined values. The standards are obtained by mixing equal volumes of different microbial standards according to different combinations and then diluting them proportionally to multiple gradients.

[0052] In some embodiments, the method for achieving the determination of the value is at least one of digital PCR determination of the value and standard determination of the value.

[0053] In some embodiments, the kit further includes a positive control and a negative control, wherein the positive control is at least one of a culture of the above-mentioned microorganism, a plasmid fragment or a DNA fragment; and the negative control is at least one of a negative sample and an internal standard plasmid.

[0054] In another aspect, the present invention provides a microecological evaluation method for rapid synchronous quantitative detection of reproductive tract microorganisms, the method comprising the following steps:

[0055] (1) Extraction of nucleic acid from samples to be tested;

[0056] (2) Preparation of reaction system: using the first primer probe combination, the second primer probe combination, the third primer probe combination, the fourth primer probe combination, the fifth primer probe combination, the sixth primer probe combination, the seventh primer probe combination, the eighth primer probe combination and the internal standard primer probe combination, respectively, together with the amplification reaction solution to prepare a reaction system;

[0057] (3) The PCR amplification procedure is as follows: 30-50°C for 1 min-10 min; 94-97°C for 10 s-10 min; 94-97°C for 5 s-15 s, 55-60°C for 10 s-60 s*, for a total of 40-45 cycles, and fluorescence is collected at *.

[0058] (4) Result analysis: The Ct value of the detection channel FAM / VIC / ROX / CY5 is ≤38.0, the Ct value of the QUASAR 705 channel is ≤37.0, and the amplification curve is S-shaped with an obvious exponential growth period, which is judged as positive for the corresponding microorganism. The quantitative detection of the sample is achieved based on the standard curve of the target microorganism DNA-CT drawn based on multiple gradient quantitative standards;

[0059] (5) Microecological evaluation: A microecological evaluation system is established to analyze the results of the Lactobacillus group and genital pathogenic microorganisms, and the microecology of the patient's genital tract is evaluated based on the different ratios of the Lactobacillus group and genital pathogenic microorganisms.

[0060] In some embodiments, the quantitative detection in step (4) refers to the quantitative detection of reproductive tract microorganisms by establishing a standard curve, which is corrected by a high-level quantitative traceability system, so that all target microorganisms can be accurately quantified.

[0061] In some embodiments, the evaluation of reproductive tract microecology in step (5) includes:

[0062] The vaginal microecology was evaluated based on the fixed value results of four vaginal lactobacilli groups, namely Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus iners; that is, when the proportion of Lactobacillus crispatus was high, the result was "normal flora, CSTI"; when the proportion of Lactobacillus jensenii was high, the result was "normal flora, CSTV"; when the proportion of Lactobacillus gasseri was high, the result was "normal flora, CSTII"; when the proportion of Lactobacillus iners was high, the result was "decreased flora function, CSTIII"; when the proportion of pathogenic microorganisms of bacterial vaginitis (BV) or aerobic vaginitis (AV) was higher than that of the lactobacilli group, the result was "dysbiosis".

[0063] In some embodiments, the evaluation of bacterial vaginosis (BV) comprises:

[0064]

[0065] Among them, if the P value (BV) ≤ 1, it is judged as positive for bacterial vaginosis, otherwise it is judged as negative for bacterial vaginosis;

[0066] In some embodiments, the evaluation of aerobic vaginitis (AV) comprises:

[0067]

[0068] Among them, if the P value (AV) ≤ 1, it is judged as aerobic bacterial vaginitis positive, otherwise it is judged as aerobic bacterial vaginitis negative.

[0069] In some embodiments, the evaluation of vulvovaginal candidiasis (VVC) includes: if the detection of Candida spp. and / or Candida albicans is positive, it is judged as VVC positive, otherwise it is judged as VVC negative;

[0070] In some embodiments, the evaluation of vaginal trichomoniasis (TV) includes: if the test result of vaginal trichomoniasis is positive, it is judged as TV positive, otherwise it is judged as TV negative;

[0071] In some embodiments, the evaluation of sexually transmitted diseases (STI) includes: if one or more of the tests for Neisseria gonorrhoeae, Treponema pallidum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus type I, and herpes simplex virus type II are positive, it is judged as STI positive, otherwise it is judged as STI negative;

[0072] In some methods, the HPV evaluation: if any one or more of HPV16, HPV18 and other high-risk HPV types are detected positive, it is judged as HPV positive, otherwise it is judged as HPV negative.

[0073] Compared with the prior art, the present invention has the following beneficial effects:

[0074] (1) It can rapidly detect nearly 50 different types and types of reproductive tract microorganisms within 30 minutes, and distinguish 32 of them, comprehensively covering common vaginal lactobacilli, most pathogenic reproductive tract microorganisms, sexually transmitted microorganisms and cervical microorganisms, and can realize rapid detection of single or mixed reproductive tract microbial infections;

[0075] (2) It has the advantages of high sensitivity, strong specificity, wide linear range, good accuracy, rapidity, convenience and high throughput;

[0076] (3) Each group has a human genome internal standard, which can be used to monitor sample collection, storage and transportation, nucleic acid extraction, and PCR amplification processes to avoid false negative results;

[0077] (4) The specific Taq enzyme modified by directed mutagenesis has the advantages of rapidity and specificity, prevents nonspecific amplification, significantly improves detection accuracy and sensitivity, and adds UDG enzyme to effectively avoid the occurrence of false positives;

[0078] (5) The reproductive tract microorganism quantification method of the present invention is calibrated by a high-level measurement system and can achieve accurate quantification of all microorganisms within the detection range;

[0079] (6) The quantitative evaluation system for reproductive tract microecology of the present invention can evaluate the reproductive tract microecology by monitoring the proportion of vaginal lactobacilli, such as determining the dominant flora, flora diversity, etc., and can quickly diagnose various vaginitis, sexually transmitted diseases, and HPV. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figures 1 to 3 The standard curves of combinations one, three, and six in Example 2;

[0081] Figure 4 The test results of simulated samples 1 to 8 in Example 3;

[0082] Figures 5 to 12 The results of the sensitivity test of the primer-probe combination of combination 1 to combination 8 in Example 3 to each target;

[0083] Fig.13 The specificity detection results of each set of primer-probe combinations in Example 3;

[0084] Fig.14 The precision test results of each set of primer-probe combinations of combination 1 to combination 4 in Example 3;

[0085] Fig.15 The precision test results of the primer-probe combinations of combinations 5 to 8 in Example 3;

[0086] Fig.16 This is a schematic diagram of the results of detection by the microecological evaluation system in Example 4. DETAILED DESCRIPTION

[0087] The present invention is further described in detail below in conjunction with the examples. It should be noted that the examples described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. The reagents used in this example are all known products and were obtained by purchasing commercially available products.

[0088] Example 1: Primer probe sequences and combinations for detecting reproductive tract microorganisms

[0089] The present invention designs specific primers and probe sequences with the conserved regions of the genomes of various reproductive tract microorganisms and internal reference genes as targets. The primer and probe sequences of each microorganism are shown in Table 4.

[0090] Table 4 Primer and probe sequences for reproductive tract microorganisms

[0091]

[0092] The present invention divides the above-mentioned reproductive tract microorganisms and internal standard genes into eight primer-probe combinations, and respectively labels the fluorescent groups FAM / VIC / ROX / CY5 / QUASAR 705 at the 5' end of the probe, and labels the quenching groups BHQ1 / BHQ2 / BHQ3 at the 3' end of the probe. The grouping method and the setting method of the fluorescent groups are shown in Table 5 below.

[0093] Table 5 Grouping method and fluorescent group setting method

[0094]

[0095] The ROX channel shown in the sixth group includes 12 groups of primer probes for HPV types numbered 23-34 in Table 4. The fluorescent groups of these 12 groups of probes all adopt ROX fluorescent groups. The same fluorescent group is used to detect 15 other high-risk types of HPV. Once one of them is positive, it can be detected.

[0096] Example 2: Method for simultaneous quantitative detection of reproductive tract microorganisms provided by the present invention

[0097] This embodiment is based on the grouping method shown in Example 1 and the setting method of the fluorescent groups of each group of probes, and uses a five-channel fluorescent quantitative PCR instrument for detection.

[0098] 1. Specific detection methods

[0099] 1. Preparation of reaction system

[0100] Prepare the amplification reaction system according to Table 5, take 8 tubes of primer-probe combination and amplification reaction solution and add them to the reagent tubes in proportion, mix them thoroughly to prepare reaction systems one to eight, centrifuge them instantaneously and set them aside.

[0101] Table 5. Reaction system formula

[0102]

[0103] The amplification reaction solution contains 3~10U / Test (preferably 8U / Test in this embodiment) of Taq DNA polymerase (purchased from Shanghai Shuoying Biotechnology Co., Ltd., model CSMix-005), 3~10U / Test (preferably 8U / Test in this embodiment) of UDG enzyme, 2×Buffer buffer, 0~10mM (preferably 5 mM in this embodiment) of MgCl2, and 0.5~1mM (preferably 1mM in this embodiment) of dNTPs (dATP: dTTP: dCTP: dGTP: dUTP=1:1:1:1:1), prepared with RNase-free water.

[0104] 2. Extraction of sample nucleic acid

[0105] This example uses clinically collected cervical / vaginal swab samples (vaginal swab samples are used in this example) and uses the nucleic acid extraction and purification kit (magnetic bead method) of Jiangsu Shuoshi Biotechnology Co., Ltd. to extract nucleic acid. Positive control, negative control and quantitative standard products are taken for nucleic acid extraction simultaneously.

[0106] 3. Add sample

[0107] Add 5 µL of the extracted positive control, negative control, quantitative standard and sample nucleic acid to the prepared reagent tubes, cover the tubes tightly and centrifuge at low speed.

[0108] 4. Amplification Reaction Procedure

[0109] Place the above reagent tubes into the SLAN-96H fluorescence PCR instrument, set the sample name, standard and its corresponding concentration to be tested, and start amplification detection. The multiplex fluorescence PCR reaction conditions are shown in Table 6.

[0110] Table 6. Amplification procedures

[0111]

[0112] 2. Results Analysis

[0113] The results are automatically interpreted by the software. The default baseline start cycle is 6, the end cycle is 12, and the threshold is 0.12. Click "Analyze" to automatically obtain the analysis results. The positive control and negative control should meet the requirements of the following "Quality Control". Quality control is as follows:

[0114] (1) Positive control: Ct values ​​of FAM, VIC, ROX, CY5, and QUASAR 705 channels ≤ 30.0; (2) Negative control: No Ct values ​​(NoCt) of FAM, VIC, ROX, and CY5 channels, and Ct value of QUASAR 705 channel ≤ 30.0.

[0115] The above requirements must be met in the same experiment, otherwise the experiment will be invalid.

[0116] The Ct value of the detection channel FAM / VIC / ROX / CY5 was ≤38.0, the Ct value of the QUASAR 705 channel was ≤37.0, and the amplification curve was S-shaped with an obvious exponential growth period, which was judged as positive for the corresponding microorganism.

[0117] 1. Quantitative analysis

[0118] (1) Establishment of standard curve

[0119] The standards of 32 target microorganisms were quantified by digital PCR. Nucleic acid protectant (0.1-10mmol / L TCEP) was used to dilute them to 5×10 9 copies / mL, and mix equal volumes of combinations 1 to 8 to obtain eight tubes of standard products S1 to S8. S1 to S8 were diluted 10 times in a series of concentrations so that the concentration of each target microorganism had four consecutive concentrations in the following concentration gradient: 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 The above standards were amplified by fluorescence quantitative PCR according to the above method, and the standard curve of each target microorganism was fitted according to the CT value and concentration Log value of the detection results of 32 target microorganism standards. This example takes combinations 1, 3, and 6 as examples, and their standard curves are shown as follows: Figure 1~Figure 3 shown.

[0120] (2) Quantification of reproductive tract microorganisms

[0121] The reproductive tract microorganisms were quantified based on the standard curves drawn with the above-mentioned different quantitative standards and the CT values ​​of the detection results of each target microorganism.

[0122] 2. Microecological evaluation

[0123] Since vaginitis is caused by mixed infection of multiple bacteria, involving a decrease in lactobacilli and an increase in anaerobic bacteria, it is necessary to comprehensively analyze the fixed value proportion of relevant target microorganisms in the sample. Based on the quantitative detection results of 32 reproductive tract microorganisms by the above detection method, a simple, fast and highly accurate microecological evaluation system was constructed, which mainly includes the evaluation of bacterial vaginitis, aerobic vaginitis, vulvovaginal candidiasis (VVC), vaginal trichomoniasis (TV), sexually transmitted diseases (STI), and HPV. The specific methods are as follows:

[0124] (1) Evaluation of reproductive tract microecology:

[0125] The vaginal microecology was evaluated based on the fixed value results of four vaginal lactobacilli groups, namely Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus iners; that is, when the proportion of Lactobacillus crispatus was high, the result was "normal flora, CSTI"; when the proportion of Lactobacillus jensenii was high, the result was "normal flora, CSTV"; when the proportion of Lactobacillus gasseri was high, the result was "normal flora, CSTII"; when the proportion of Lactobacillus iners was high, the result was "decreased flora function, CSTIII"; when the proportion of pathogenic microorganisms of bacterial vaginitis (BV) or aerobic vaginitis (AV) was higher than that of the lactobacilli group, the result was "dysbiosis".

[0126] (2) Evaluation of reproductive tract diseases

[0127] 1) Evaluation of bacterial vaginosis (BV):

[0128]

[0129] Among them, if the P value (BV) ≤ 1, it is judged as positive for bacterial vaginosis, otherwise it is judged as negative for bacterial vaginosis;

[0130] 2) Evaluation of aerobic vaginitis (AV):

[0131]

[0132] Among them, if the P value (AV) is ≤1, it is judged as positive for aerobic bacterial vaginitis, otherwise it is judged as negative for aerobic bacterial vaginitis;

[0133] 3) Evaluation of vulvovaginal candidiasis (VVC): If the test result of Candida spp. and / or Candida albicans is positive, the patient is considered VVC positive; otherwise, the patient is considered VVC negative;

[0134] 4) Evaluation of vaginal trichomoniasis (TV): If the test result of vaginal trichomoniasis is positive, it is judged as TV positive, otherwise it is judged as TV negative;

[0135] 5) Evaluation of sexually transmitted diseases (STI): If one or more of the tests for Neisseria gonorrhoeae, Treponema pallidum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus type I, or herpes simplex virus type II are positive, then the patient is considered STI positive; otherwise, the patient is considered STI negative.

[0136] 6) HPV evaluation: If any one or more of HPV16, HPV18 and other high-risk HPV types are tested positive, it is judged as HPV positive, otherwise it is judged as HPV negative.

[0137] Example 3: Simulated sample detection and performance analysis

[0138] 1. Simulated sample test results

[0139] The simulated samples 1 to 8 used for detection were: simulated sample 1 was a mixture of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, Lactobacillus iners and an internal standard plasmid; simulated sample 2 was a mixture of Atopobium vaginalis, Prevotella, Mycoplasma genitalium, Mycoplasma hominis and an internal standard plasmid; simulated sample 3 was a mixture of Gardnerella vaginalis, Bacteroides, Mobiluncus, herpes simplex virus type 2 and an internal standard plasmid; simulated sample 4 was a mixture of Candida albicans, Candida tropicalis, Escherichia coli, Staphylococcus aureus and an internal standard plasmid. Simulated sample 5 is a mixture of Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Trichomonas vaginalis and internal standard plasmid; simulated sample 6 is a mixture of HPV16, HPV18, other high-risk HPV, group B Streptococcus and internal standard plasmid; simulated sample 7 is a mixture of Megasphaera, BVAB2, Treponema pallidum, herpes simplex virus type 1 and internal standard plasmid; simulated sample 8 is a mixture of Streptococcus pharyngitis, Enterococcus faecalis, Klebsiella pneumoniae, group A Streptococcus and internal standard plasmid. The content of each microorganism in the simulated samples is prepared separately according to needs. The test results of simulated samples 1 to 8 are as follows Figure 4 As shown, the results show that the detection method provided in this embodiment can well distinguish the microorganisms within the detection range.

[0140] 2. Sensitivity analysis

[0141] The sensitivity of each target was tested using the primer-probe combination in Table 4. The clinical samples with fixed values ​​were diluted to 2000, 1000, 500, 250, and 100 copies / mL, and each was tested 3 times. The lowest concentration detected was tested 20 times. The results are as follows: Figure 5~Figure 12 As shown, the results show that the composition can detect samples with a concentration of 100 to 2000 copies / mL, indicating that the sensitivity of the composition of the present invention to different target microorganisms is 100 copies / mL.

[0142] 3. Specificity analysis

[0143] Sequence comparison analysis was performed on the microbial target genes within the detection range of the primer-probe combination in Table 4 and pathogens outside the detection range with the same infection site or similar symptoms (BVAB1, varicella-zoster virus, HPV6, HPV11, Acinetobacter baumannii, Pseudomonas aeruginosa, Mycobacterium tuberculosis, cytomegalovirus), and the kit was used to detect high-concentration pathogen samples. The results are shown in FIG. Fig.13 , showing that the kit could not detect these pathogens, indicating that the composition of the present invention has good specificity.

[0144] 4. Precision analysis

[0145] This example uses the simulated samples 1 to 8 described above, and uses the above method to perform 10 repeated tests. The test results are shown in Figure 14~Figure 15 The coefficient of variation (CV, %) of the calculated Ct values ​​is shown in Table 7. The results show that the CV values ​​are all <1.74%, indicating that the precision of the composition is good.

[0146] Table 7. Precision results

[0147]

[0148] Example 3: Accuracy Verification

[0149] 1. Comparison with the gold standard method

[0150] 25 clinical samples diagnosed by gold standard (Nugent comprehensive scoring method) or microscopic observation were collected from the hospital, including 10 positive samples diagnosed as bacterial vaginosis (BV), 7 positive samples of vulvovaginal candidiasis (VVC), 3 positive samples of vaginal trichomoniasis (TV), and 5 normal samples. The present composition was used for testing to verify the accuracy of the application of the present composition. The test comparison results are shown in Table 13. This embodiment takes Sample 1, Sample 11, Sample 18, and Sample 21 as examples, and the test results are shown in Table 13. Fig.16 shown.

[0151] Table 13. Comparison results between the present composition and the gold standard method

[0152]

[0153] It can be seen from Table 13 that the microecological evaluation method provided in this embodiment is consistent with the results of the gold standard method, and compared with the gold standard, the method provided in this embodiment can also provide more detailed and accurate microecological flora evaluation results, which is conducive to doctors directly providing more targeted diagnosis and treatment plans.

[0154] 2. Comparison with tNGS method

[0155] Seven clinical samples were collected from the hospital and tested simultaneously using the tNGS method and the composition of the present invention to verify the accuracy of the composition of the present invention. The results are shown in Table 14 below.

[0156] Table 14. Comparison results between the present composition and the tNGS method

[0157]

[0158] The results of Table 14 show that the results of the present invention are consistent with those of the tNGS method. Moreover, compared with the tNGS method, the method provided in this embodiment is convenient to operate, low in cost, can cover the main reproductive tract microorganisms, and quantify each microorganism, which has obvious advantages.

[0159] The above results show that the present invention can quickly provide evaluation results that are completely consistent with the gold standard and tNGS method by quickly and synchronously quantitatively detecting up to 50 different types and types of reproductive tract microorganisms, combined with the microbial evaluation method provided by the microorganisms provided by the present invention, making the microbial evaluation process simpler, more convenient and more efficient.

[0160] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A primer-probe combination for rapid and synchronous quantitative detection of reproductive tract microorganisms, characterized in that: Including any one or more of the 32 sets of primer-probe combinations shown in the following table: 。 2. The primer-probe combination according to claim 1, characterized in that: It includes eight sets of primer-probe combinations as shown in the following table, each of which can achieve synchronous detection of reproductive tract microorganisms within the group: 。 3. The primer-probe combination according to claim 2, characterized in that: The sixth group also includes any one or more of the 11 primer-probe combinations shown in the following table: 。 4. A kit for rapid and synchronous detection of reproductive tract microorganisms, characterized in that: Comprising the primer-probe combination as described in any one of claims 2 to 3.

5. The kit according to claim 4, characterized in that The method also includes an amplification reaction solution, which includes Taq DNA polymerase and UDG enzyme.

6. The kit according to claim 5, characterized in that The amplification reaction solution also includes PCR buffer, MgCl2, dNTPs and RNase-free water.

7. A quantitative evaluation system for rapid and synchronous detection of reproductive tract microecology, characterized in that: It comprises the primer-probe combination as described in any one of claims 2 to 3, the kit as described in any one of claims 4 to 6, and a microecological evaluation method.

8. The quantitative evaluation system according to claim 7, characterized in that: The microecological evaluation method includes: evaluation of reproductive tract microecology: evaluating vaginal microecology according to the fixed value results of four vaginal lactobacilli groups, namely, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners; that is, when the proportion of Lactobacillus crispatus is high, the result is "normal flora, CSTI"; when the proportion of Lactobacillus jensenii is high, the result is "normal flora, CSTV"; when the proportion of Lactobacillus gasseri is high, the result is "normal flora, CSTII"; when the proportion of Lactobacillus iners is high, the result is "decreased flora function, CSTIII"; when the proportion of pathogenic microorganisms of bacterial vaginitis (BV) or aerobic bacterial vaginitis (AV) is higher than that of the lactobacilli group, the result is "dysbiosis"; the evaluation of bacterial vaginitis (BV) includes: If the P value (BV) is ≤1, it is considered positive for bacterial vaginosis, otherwise it is considered negative for bacterial vaginosis. The evaluation of aerobic vaginitis (AV) includes: Among them, if the P value (AV) is ≤1, it is judged as aerobic vaginitis positive, otherwise it is judged as aerobic vaginitis negative; evaluation of vulvovaginal candidiasis (VVC): if the test results of Candida spp. and / or Candida albicans are positive, it is judged as VVC positive, otherwise it is judged as VVC negative; evaluation of vaginal trichomoniasis (TV): if the test results of Trichomonas vaginalis are positive, it is judged as TV positive, otherwise it is judged as TV negative; evaluation of sexually transmitted diseases (STI) includes: if one or more of the tests of Neisseria gonorrhoeae, Treponema pallidum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus type I, and herpes simplex virus type II are positive, it is judged as STI positive, otherwise it is judged as STI negative; evaluation of HPV: if any one or more of the tests of HPV16, HPV18, and other high-risk HPV types are positive, it is judged as HPV positive, otherwise it is judged as HPV negative.

9. A microecological evaluation system for rapid and synchronous quantitative detection of reproductive tract microorganisms, characterized in that: The system performs quantitative detection and microecological evaluation of nearly 50 different types and types of reproductive tract microorganisms in the sample based on a standard curve of target microbial DNA-CT drawn with multiple gradient quantitative standards, thereby realizing auxiliary diagnosis of various reproductive tract infections; the quantitative detection results of the microorganisms in the sample are obtained by multiple PCR amplification and detection using the primer-probe combination described in any one of claims 2 to 3.

10. The microecological evaluation system according to claim 9, characterized in that: The system uses the following evaluation method for evaluation and analysis, and the evaluation method includes: evaluation of reproductive tract microecology: evaluating vaginal microecology according to the fixed value results of four vaginal lactobacillus groups, namely, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners; that is, when the proportion of Lactobacillus crispatus is high, the result is "normal flora, CSTI"; when the proportion of Lactobacillus jensenii is high, the result is "normal flora, CSTV"; when the proportion of Lactobacillus gasseri is high, the result is "normal flora, CSTII"; when the proportion of Lactobacillus iners is high, the result is "decreased flora function, CSTIII"; when the proportion of pathogenic microorganisms of bacterial vaginitis (BV) or aerobic bacterial vaginitis (AV) is higher than that of the lactobacillus group, the result is "dysbiosis"; the evaluation of bacterial vaginitis (BV) includes: If the P value (BV) is ≤1, it is considered positive for bacterial vaginosis, otherwise it is considered negative for bacterial vaginosis. The evaluation of aerobic vaginitis (AV) includes: Among them, if the P value (AV) is ≤1, it is judged as aerobic vaginitis positive, otherwise it is judged as aerobic vaginitis negative; evaluation of vulvovaginal candidiasis (VVC): if the test results of Candida spp. and / or Candida albicans are positive, it is judged as VVC positive, otherwise it is judged as VVC negative; evaluation of vaginal trichomoniasis (TV): if the test results of Trichomonas vaginalis are positive, it is judged as TV positive, otherwise it is judged as TV negative; evaluation of sexually transmitted diseases (STI) includes: if one or more of the tests of Neisseria gonorrhoeae, Treponema pallidum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus type I, and herpes simplex virus type II are positive, it is judged as STI positive, otherwise it is judged as STI negative; evaluation of HPV: if any one or more of the tests of HPV16, HPV18, and other high-risk HPV types are positive, it is judged as HPV positive, otherwise it is judged as HPV negative.

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