A primer-probe combination, kit, and microecological evaluation method for rapid and simultaneous quantitative detection of reproductive tract microorganisms

Abstract

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. By designing the primer-probe combination and improving the amplification reaction conditions, the synchronous quantitative detection of nearly 50 different types and types of microorganisms in the genital tract can be achieved, covering vaginal lactobacilli, pathogenic genital tract microorganisms, sexually transmitted microorganisms, and cervical microorganisms. 32 of these microorganisms can be distinguished, achieving comprehensive coverage of the main microorganisms in the genital tract. At the same time, corresponding microecological evaluation methods are also designed to enable rapid diagnosis of various vaginitis, sexually transmitted diseases, and HPV, providing a basis for the clinical treatment and prognosis of genital tract infections.

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 and synchronous quantitative detection of reproductive tract microorganisms. Background Art

[0002] Reproductive tract infections (RTIs) are common gynecological conditions in women and a global social and public health concern. Common RTIs include vaginitis (bacterial, trichomonal, candidiasis, and aerobic vaginitis), sexually transmitted diseases (STDs), and cervical diseases. The primary cause of RTI is invasion by a variety of microorganisms, including bacteria, viruses, mycoplasmas, fungi, and Trichomonas. A decrease or absence of Lactobacilli (Lactobacillus crispatus, Lactobacillus agar, Lactobacillus jennis, and Lactobacillus iners), which are dominant in the normal vaginal flora, weakens their inhibitory effect on other microorganisms, allowing pathogens to proliferate, leading to vaginitis. Bacterial vaginitis, aerobic vaginitis, Trichomonas vaginitis and candidiasis 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 causing bacterial vaginosis include Gardnerella vaginalis, Atopobium vaginalis, Prevotella, Mobiluncus, Bacteroides, Megasphaera, bacterial vaginosis-associated bacteria 2 (BVAB2), Ureaplasma urealyticum, Mycoplasma hominis, etc.; the microorganisms causing aerobic vaginitis mainly include group A Streptococcus, group B Streptococcus, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Streptococcus anginosus, and Klebsiella pneumoniae; the microorganism causing trichomonas vaginitis is Trichomonas vaginalis; vulvovaginal candidiasis, also known as vulvovaginal candidiasis, is a reproductive tract disease caused by Candida infection. The main pathogen is Candida albicans, and other non-albicans such as Candida glabrata, Candida tropicalis, and Candida krusei account for a minority. Sexually transmitted diseases (STDs) are widespread, with gonorrhea being the most common. Neisseria gonorrhoeae is the primary cause. Chlamydia trachomatis is a common cause of non-gonococcal urethritis, and Mycoplasma genitalium can also cause non-gonococcal urethritis. Treponema pallidum is the microorganism that causes syphilis, and over 95% of syphilis infections are transmitted through sexual contact. Herpes simplex virus types 1 and 2 can cause vesicular infections in the genital and perianal areas, leading to STDs. There are over 200 subtypes of human papillomavirus (HPV) that can infect humans, over 40 of which can cause disease, primarily through contact. Persistent HPV infection can cause proliferative changes in the genital mucosa and skin, clinically manifesting as various types of epithelial warts and malignant tumors such as cervical, anal, and vaginal cancer. The World Health Organization (WHO) categorizes HPV into 14 high-risk types based on their severity: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68; four intermediate-high-risk types: HPV 26, 53, 73, and 82; and the remaining low-risk types. Persistent infection with high-risk HPV types is the leading cause of cervical cancer and its precancerous lesions.

[0004] The above microorganisms may infect alone or in combination with multiple microorganisms. Mixed infections of reproductive tract microorganisms are often accompanied by a complex reproductive tract environment. Compared with single microbial infections, the diagnosis and treatment of mixed infections are more difficult.

[0005] Clinically, vaginal microecological testing primarily involves morphological and functional testing. Traditional morphological and functional testing, culture methods, and serological testing methods are low-cost and easy to use, but rely primarily on microscopic observation and judgment. This requires significant manpower and the skills and experience of the tester. These methods are unable to specifically identify and quantify microorganisms, and are incapable of comprehensive and rapid diagnosis of genital tract infections caused by multiple microorganisms. Given the time required to test for mixed genital tract microbial infections, clinical practice requires methods that can quickly and accurately detect the primary microorganisms causing genital tract infections and diagnose the genital tract microecological status based on 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 reagents currently available on the market for detecting genital tract infection microorganisms based on molecular diagnostic technology are time-consuming, and most of them are qualitative detection of a single microorganism or several microorganisms that cause a single vaginitis, and cannot simultaneously cover the main microorganisms of multiple genital tract infections. Moreover, there are currently no products with rapid quantitative detection and microecological evaluation functions. For example, the primer-probe combination provided by CN117925870A can simultaneously detect 11 vaginal microorganisms, including Gardnerella vaginalis, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Group B Streptococcus, Trichomonas vaginalis, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus crispatus. Detection is performed using a five-fluorescence channel PCR detection device. However, since it can only detect 11 microorganisms simultaneously, 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. This method is of great significance for the realization of multiple joint detection of reproductive tract microorganisms and the study of sexually transmitted diseases. Summary of the Invention

[0008] In response to 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 lactobacilli, 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, corresponding microecological evaluation methods are 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 quantitatively detects reproductive tract microorganisms based on a standard curve established after multiple PCR amplification using a variety of primer and probe combinations, and is corrected by a high-level quantity 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 supporting evaluation methods to evaluate and analyze the reproductive tract microbiome. This includes determining the status of the reproductive tract flora and rapidly diagnosing reproductive tract diseases, including bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, 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 proportions of lactobacilli 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 vaginosis and aerobic vaginitis by calculating the fixed value ratio of the 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 uses a method of synchronously monitoring the dominant vaginal flora and the main microorganisms causing reproductive tract infections to assist in the diagnosis of the patient's reproductive tract microecological status and provide a basis for the clinical treatment and prognosis of reproductive tract infections. It covers:

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

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

[0020] 3. Seven 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 vaginosis. Among them, Group B Streptococcus, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis are the species with the highest clinical incidence;

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

[0022] 5. Four candidiasis microorganisms (Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei, of which only Candida albicans can be typed) cover more than 90% of clinical candidiasis 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 common sexually transmitted disease microorganisms.

[0024] The present invention designs specific amplification primers and Taqman probes based on the internal standard fragments 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 or negative nature of the target genes of the reproductive tract microorganisms.

[0025] Currently, commercially available fluorescence quantitative PCR instruments typically have 4 to 6 fluorescence channels, so multiplex fluorescence quantitative PCR can detect 3-5 pathogens and 1 internal standard gene in a single system. Therefore, we grouped the 32 microorganisms for testing, selecting 3-5 microorganisms from each group and assigning them to separate sample tubes for simultaneous amplification and quantitative detection. Following the principles of multiplex primer-probe design, the primer pairs and probes for the 3-5 microorganisms in the same group were labeled with different fluorescent groups and detected using different channels, preventing interference.

[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] By adding a human genome internal standard, the present invention can monitor the sample collection, storage and transportation, nucleic acid extraction and PCR amplification processes, 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. 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 other high-risk HPV typing primer-probe combinations shown in Table 3:

[0034] Table 3. Primer and 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 for different HPV types can be shared, such as HPV31 and HPV35, HPV56 and HPV66, HPV39 and HPV68, HPV33 and HPV58, and HPV51 and HPV82.

[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 quencher 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, comprising the primer-probe combination 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-contamination 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 utilizes a specific Taq DNA polymerase engineered through targeted mutagenesis (Thermo Scientific™ DreamTaq™ DNA polymerase is an enhanced version of Taq DNA polymerase), which helps improve detection accuracy and sensitivity. This may be due to the fact that this targeted mutagenesis-engineered specific Taq DNA polymerase has stricter priming requirements than conventional Taq enzymes. Furthermore, the enzyme antibody has been enhanced in purity and concentration, effectively blocking Taq enzyme activity. Furthermore, the enzyme antibody also utilizes a monoclonal antibody targeting the active center region, resulting in more robust binding, effectively preventing nonspecific amplification and interference between different primers and 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 standards for quantitative detection, and the quantitative standards are WHO standards, national quantitative standards, third-party quantitative standards, or target microbial sample nucleic acids, pseudovirus nucleic acids, or plasmid DNA with a fixed value. The standards are obtained by mixing equal volumes of different microbial standards in 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 and synchronous quantitative detection of reproductive tract microorganisms, the method comprising the following steps:

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

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

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

[0058] (4) Result analysis: The Ct value of the detection channel FAM / VIC / ROX / CY5 is ≤38.0, and the Ct value of the QUASAR 705 channel is ≤37.0. The amplification curve is S-shaped with a clear exponential growth period, which is determined to be positive for the corresponding microorganism. 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 determination of Lactobacillus groups and genital pathogenic microorganisms respectively, and the microecology of the patient's genital tract is evaluated based on the different ratios of Lactobacillus groups and genital pathogenic microorganisms.

[0060] In some embodiments, the quantitative detection described 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 the reproductive tract microecology in step (5) includes:

[0062] The vaginal microecology was assessed based on the fixed values ​​of four vaginal lactobacilli groups: 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 vaginosis (BV) or aerobic vaginitis (AV) was higher than that of the lactobacilli group, the result was "dysbacteriosis".

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

[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) includes:

[0067]

[0068] Among them, if the P value (AV) ≤ 1, it is judged as aerobic bacterial vaginosis positive, otherwise it is judged as aerobic bacterial vaginosis 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 Trichomonas vaginalis (TV) includes: if the test result of Trichomonas vaginalis 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 (STIs) includes: if one or more of the tests for Neisseria gonorrhoeae, Treponema pallidum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus type 1, and herpes simplex virus type 2 are positive, the test is considered STI positive; otherwise, the test is considered STI negative.

[0072] In some methods, the 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.

[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 the 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 is equipped with a human genome internal standard, which can be used to monitor the sample collection, storage and transportation, nucleic acid extraction and PCR amplification process, thus avoiding 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 microbial quantification method of the present invention is calibrated by a high-level measurement system and can accurately quantify all microorganisms within the detection range;

[0079] (6) The quantitative evaluation system of the 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 and flora diversity, and can quickly diagnose various vaginitis, sexually transmitted diseases, and HPV. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figures 1-3 The standard curves for combinations 1, 3, and 6 in Example 2 are shown;

[0081] Figure 4 The test results of simulated samples 1 to 8 in Example 3 are shown below:

[0082] Figures 5 to 12 The results of the sensitivity test of the primer-probe combinations of combinations 1 to 8 to various targets in Example 3 are shown;

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

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

[0085] Figure 15 The precision test results of each primer-probe combination of combination 5 to combination 8 in Example 3;

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

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

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

[0089] The present invention designs specific primers and probe sequences targeting the conserved regions of the genomes of various reproductive tract microorganisms and internal reference genes. 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 labels the 5' end of the probe with the fluorescent group FAM / VIC / ROX / CY5 / QUASAR 705, and the 3' end of the probe with the quencher group BHQ1 / BHQ2 / BHQ3. The grouping method and the setting method of the fluorescent group 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 use 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 and the setting method of the fluorescent groups of each group of probes shown in Example 1, 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 thoroughly to prepare reaction systems 1 to 8. Centrifuge briefly and set aside.

[0101] Table 5. Reaction system formula

[0102]

[0103] The amplification reaction solution contains 3-10 U / Test (preferably 8 U / Test in this embodiment) of Taq DNA polymerase (purchased from Shanghai Shuoying Biotechnology Co., Ltd., model CSMix-005), 3-10 U / Test (preferably 8 U / Test in this embodiment) of UDG enzyme, 2× Buffer, 0-10 mM (preferably 5 mM in this embodiment) of MgCl2, and 0.5-1 mM (preferably 1 mM 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 (in this example, vaginal swab samples) and uses a nucleic acid extraction and purification kit (magnetic bead method) from Jiangsu Shuoshi Biotechnology Co., Ltd. for nucleic acid extraction. Positive and negative controls and quantitative standards are also extracted simultaneously.

[0106] 3. Add sample

[0107] Add 5µL of the extracted positive control, negative control, quantitative standard, and sample nucleic acid to the prepared reagent tubes, cap the tubes tightly, and centrifuge briefly 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 corresponding concentration to be tested, and begin amplification. 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 and negative controls should meet the requirements of the "Quality Control" below. 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 simultaneously 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, and the Ct value of the QUASAR 705 channel was ≤37.0. The amplification curve was S-shaped with a clear exponential growth period, and the corresponding microorganism was determined to be positive.

[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 (0.1-10 mmol / L TCEP) was used to dilute them to 5×10 9 10 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-fold to make the concentration of each target microorganism have 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. Based on the CT value and concentration Log value of the detection results of 32 target microorganism standards, the standard curve of each target microorganism was fitted. This example uses combinations 1, 3, and 6 as examples. Their standard curves are as follows: Figures 1 to 3 shown.

[0120] (2) Quantification of reproductive tract microorganisms

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

[0122] 2. Microecological evaluation

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

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

[0125] The vaginal microecology was assessed based on the fixed values ​​of four vaginal lactobacilli groups: 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 vaginosis (BV) or aerobic vaginitis (AV) was higher than that of the lactobacilli group, the result was "dysbacteriosis".

[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) ≤ 1, it is judged as aerobic vaginitis positive, otherwise it is judged as aerobic vaginitis negative;

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

[0134] 4) Evaluation of Trichomonas vaginalis (TV): If the test result of Trichomonas vaginalis is positive, it is considered TV positive; otherwise, it is considered TV negative;

[0135] 5) Sexually Transmitted Infection (STI) Assessment: If one or more of the tests for Neisseria gonorrhoeae, Treponema pallidum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus type 1, or herpes simplex virus type 2 are positive, 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 test positive, the test is considered HPV positive; otherwise, it is considered 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 testing were as follows: 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. The simulated sample 5 is a mixture of Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Trichomonas vaginalis and an internal standard plasmid; the simulated sample 6 is a mixture of HPV16, HPV18, other high-risk HPV types, group B Streptococcus and an internal standard plasmid; the simulated sample 7 is a mixture of Megasphaera, BVAB2, Treponema pallidum, herpes simplex virus type 1 and an internal standard plasmid; the simulated sample 8 is a mixture of Streptococcus pharyngitis, Enterococcus faecalis, Klebsiella pneumoniae, group A Streptococcus and an internal standard plasmid. The content of each microorganism in the simulated sample 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 repeated 3 times. The lowest concentration detected was repeated 20 times. The results are as follows: Figures 5 to 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 alignment analysis was performed on the microbial target genes within the detection range of the primer-probe combination in Table 4 with pathogens outside the detection range that have the same infection site or cause similar symptoms (BVAB1, varicella-zoster virus, HPV6, HPV11, Acinetobacter baumannii, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and cytomegalovirus). The kit was used to detect high-concentration pathogen samples. The results are shown in Figure 4. Figure 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 above-mentioned simulated samples 1 to 8, and uses the above-mentioned method to perform 10 repeated tests. The test results are shown in FIG. Figure 14-15 The coefficient of variation (CV, %) of the calculated Ct values ​​is shown in Table 7. The results show that the CV values ​​are all less than 1.74%, indicating that the precision of this 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 were collected from the hospital and diagnosed by the gold standard (Nugent comprehensive scoring method) or microscopic observation, including 10 samples diagnosed as bacterial vaginosis (BV), 7 samples diagnosed as vulvovaginal candidiasis (VVC), 3 samples diagnosed as vaginal trichomoniasis (TV), and 5 normal samples. The present composition was used for testing to verify the accuracy of the present composition. The test comparison results are shown in Table 13. This example uses Sample 1, Sample 11, Sample 18, and Sample 21 as examples. The test results are shown in Table 13. Figure 16 shown.

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

[0152]

[0153] As can be seen from Table 13, the microecological evaluation method provided in this embodiment is consistent with the results of the gold standard method. Moreover, 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 in Table 14 show that the results of the present invention are consistent with those of the tNGS method. Furthermore, compared with the tNGS method, the method provided in this example is convenient to operate, low-cost, covers the main reproductive tract microorganisms, and quantifies 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 scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A primer-probe combination for rapid and simultaneous quantitative detection of reproductive tract microorganisms, characterized in that: It includes eight sets of primer-probe combinations as shown in the table below, each of which can achieve simultaneous detection of reproductive tract microorganisms within the group: ； The sixth group also includes any one or more of the 11 primer-probe combinations shown in the following table: ； Each of the eight primer-probe combinations further includes an internal standard primer-probe combination; 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; The 11 primer-probe combinations and the HPV31 type in the sixth group are in one channel.

2. A kit for rapid and synchronous detection of reproductive tract microorganisms, characterized in that: The method comprises the primer-probe combination as claimed in claim 1.

3. The kit according to claim 2, wherein The method also includes an amplification reaction solution, which includes Taq DNA polymerase and UDG enzyme.

4. The kit according to claim 3, wherein The amplification reaction solution also includes PCR buffer, MgCl2, dNTPs and RNase-free water.

5. A quantitative evaluation system for rapid and synchronous detection of reproductive tract microecology, characterized in that: The method comprises the primer-probe combination according to claim 1 or the kit according to any one of claims 2 to 4.

6. The quantitative evaluation system according to claim 5, wherein: The kit also includes a microecological evaluation system for performing a microecological evaluation on the test results of the kit. The microecological evaluation includes: Evaluation of the reproductive tract microbiome: The vaginal microbiome was assessed based on the concentration of four vaginal lactobacilli: Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners. Specifically, when Lactobacillus crispatus accounts for a high percentage, the result is "normal flora, CSTI"; when Lactobacillus jensenii accounts for a high percentage, the result is "normal flora, CSTV"; when Lactobacillus gasseri accounts for a high percentage, the result is "normal flora, CSTII"; and when Lactobacillus iners accounts for a high percentage, the result is "decreased flora function, CSTIII"; when the pathogenic microorganisms of bacterial vaginosis (BV) or aerobic vaginitis (AV) account for a higher percentage than the Lactobacillus group, the result is "dysbiosis"; Evaluation of bacterial vaginosis (BV) includes: ； 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; Evaluation of aerobic vaginitis (AV) includes: ； Among them, if the P value (AV) ≤ 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 for Candida spp. and / or Candida albicans are positive, the patient is considered VVC positive; otherwise, the patient is considered VVC negative. Evaluation of vaginal trichomoniasis (TV): If the test result of Trichomonas vaginalis is positive, it is judged as TV positive, otherwise it is judged as TV negative; The evaluation of sexually transmitted diseases (STIs) includes: if one or more of the tests for Neisseria gonorrhoeae, Treponema pallidum, Chlamydia trachomatis, Mycoplasma genitalium, herpes simplex virus type 1, or herpes simplex virus type 2 are positive, the patient is considered STI positive; otherwise, the patient is considered STI negative. HPV evaluation: If any one or more of HPV16, HPV18 and other high-risk HPV types test positive, it is judged as HPV positive; otherwise, it is judged as HPV negative.

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