Kit and method for simultaneously detecting 11 pathogens of urogenital system and application

Abstract

The invention provides a kit and a method for simultaneously detecting 11 pathogens of a urogenital system and application, and belongs to the field of molecular biology. The primer group is used for detecting urogenital tract pathogens, and the urogenital tract pathogens comprise ureaplasma urealyticum (UU), chlamydia trachomatis (CT), mycoplasma genitalium (MG), ureaplasma parvum (UP), mycoplasma hominis (MH), adenovirus (ADV), trichomonas vaginalis (TV), herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), haemophilus influenzae (HI) and neisseria gonorrhoeae (NG). The primer group is used for multi-joint detection of non-gonococcal urethritis related pathogens, has high specificity and sensitivity, is adaptive to non-invasive urine specimens, and supports home self-sampling. The kit is used for clinical diagnosis and can quickly judge pathogen types. Precise medication is performed based on a detection result, the healing time of a patient is shortened, the burden of a doctor is relieved, and meanwhile the pathogen transmission risk is reduced.

Description

Technical Field

[0001] The present application relates to the field of molecular biology, and specifically, to a kit, method and application for simultaneously detecting 11 urogenital pathogens, and more specifically, to a primer set and its use, a kit and its use, and a method for detecting urogenital pathogens. Background Art

[0002] Non-gonococcal urogenital tract infection, the most common is non-gonococcal urethritis (NGU). NGU refers to urethritis caused by infection with pathogens other than Neisseria gonorrhoeae (NG). Males mainly present with urethral irritation symptoms, but not as typical as gonorrhea, mainly manifested as urinary discomfort, accompanied by increased urethral secretions, which are serous or serous-purulent. Females mainly present with abnormal leucorrhea (changes in quantity, color, and taste), mainly manifested as non-gonococcal urethritis, vaginitis, cervicitis, pelvic inflammatory disease, endometritis, etc.

[0003] Chlamydia trachomatis (CT) is the most common pathogen of NGU. In recent years, Mycoplasma genitalium (MG) has also been confirmed to be the pathogen of NGU second only to CT. In addition, there are Ureaplasma urealyticum (UU), Ureaplasma par vum (UP), Mycoplasma hominis (MH), adenoviruses (ADV), Trichomonas vaginalis (TV), herpes simplex virus (HSV), Haemophilus influenzae (HI), etc.

[0004] Epidemiological studies have shown that CT, MG, UU, and MH are the most common pathogens of NGU. Among them, the infection rate of CT is 11%-50%, MG is 6%-50%, UU is 11%-26%, and MH is 1%-4.1%. There are various NGU pathogens, and there are also large differences in detection methods. Commonly used detection methods include wet mount direct microscopy, culture, immunological testing, and molecular biology testing. As the proportion of mixed infections in NGU continues to increase, the disadvantages of the above-mentioned detection methods are becoming increasingly apparent. The prolonged cure time of patients with mixed infections will cause the patients to suffer longer and the workload of doctors to increase exponentially. More importantly, it will cause the spread of NGU infection pathogens, especially drug-resistant strains.

[0005] The treatment of NGU caused by different pathogens varies significantly, so accurate detection of pathogens is of great significance for effective treatment. What clinicians need more is a combination test that covers most of the pathogens of NGU, so as to determine the precise medication after infection with the pathogen, especially as the proportion of mixed infections of NGU continues to increase, which can shorten the patient's cure time. Currently, most of the NGU pathogen detection kits on the market are based on a single or a few pathogens, which are difficult to meet the diagnostic needs of mixed infections.

[0006] The symptoms of NGU caused by different pathogens are similar, but the treatment of different pathogens is very different. Tetracyclines are generally the first choice for CT, MG, and UU infections, and macrolides are the second choice. Because MH carries the tetracycline-resistant tetM gene in the genome, the use of tetracycline is limited, and MH is naturally resistant to 14- and 15-ring macrolide antibiotics. Currently, quinolones are mainly used to treat MH infections in clinical practice. Ceftriaxone is the first choice for NG treatment, and spectinomycin is the second choice. TV infections are generally treated with metronidazole. HSV and ADV infections are generally treated as viral infections, and quinolones and macrolides are the first choice for HI infections. What clinicians need more is a combination test that covers most of the pathogens of NGU, so as to determine the right medicine after the infection, especially when the proportion of mixed infections of NGU continues to increase, which can shorten the patient's recovery time.

[0007] The NGU pathogen DNA detection kits that have been launched on the market all use male urethral secretions or female vaginal secretions as test specimens. The sampling of the above specimens requires professional personnel and may cause trauma. NGU is a sexually transmitted disease. There are cases where patients are reluctant to seek medical treatment for the sake of privacy protection, which delays the best treatment time for patients and leads to the spread of pathogens. Summary of the invention

[0008] This application aims to solve the problems of the prior art. To this end, the inventors designed primer probes for the gene-specific conserved sequences of the nine NGU infection pathogens mentioned in the 2022 edition of the "Guidelines for the Diagnosis and Treatment of Non-gonococcal Urethritis", including Ureaplasma urealyticum, Chlamydia trachomatis, Mycoplasma genitalium, Ureaplasma parvum, Mycoplasma hominis, adenovirus, Trichomonas vaginalis, herpes simplex virus (including HSV-I and HSV-II), and Haemophilus influenzae. At the same time, primer probes were designed for the gene-specific conserved sequences of Neisseria gonorrhoeae, and a real-time fluorescence PCR detection method was provided to quickly detect the genes of 11 pathogens at low cost, and nucleic acids extracted from urine and urethral (vaginal) secretion samples can be detected. It solves the problems of long culture cycle, poor sensitivity of antigen method, and low coverage and easy missed detection of single nucleic acid detection in multiple infection cases in the current detection methods of sexually transmitted disease pathogens. It is a detection product urgently needed in clinical practice.

[0009] Since NGU is a general term for diseases with similar symptoms caused by different types of pathogens such as mycoplasma, chlamydia, virus, bacteria, and Trichomonas, and the multiple infection rate is increasing, the single nucleic acid test has low coverage and is prone to missed detection. Given the high genetic homology between pathogens, especially pathogens of the same type, and the fact that there are many repetitive fragments in the gene sequence, GC content deviation, and few specific short fragments, and the serious mutual interference between specific long fragments, for example, the symptoms of diseases caused by Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium are similar, and the three mycoplasmas have certain sequence similarities, so when mixed samples obtained from patients with co-infections are amplified by PCR, mutual interference will occur, which can easily lead to incorrect diagnostic results, so there are fewer products on the market.

[0010] In addition, super-multiplex PCR detection kits are prone to the following defects: 1) Due to the genetic homology and the presence of many repetitive fragments in gene sequences among different pathogens, non-specific amplification interference is prone to occur during PCR amplification; 2) The specificity of different pathogens leads to different amplification efficiencies, and ultimately the sensitivity of detecting different pathogens varies; 3) Due to the preference of different detection channels of PCR instruments, differences in fluorescence values ​​and sensitivity will occur.

[0011] In response to defect 1), the present application provides a combination of primers and probes that do not interfere with each other and synchronously detect 11 non-gonococcal urethritis infection pathogens. The required sequence files are downloaded from public databases such as NCBI and ensembl, and the homology of the pathogen target gene is analyzed by sequence comparison using tools such as BLAST. It is found that the sequence homology of the Ure encoding gene of Ureaplasma urealyticum and Ureaplasma microphylla is 93.7%; the sequence homology of the US encoding gene of herpes simplex virus type I and type II is 50.0%; the adenovirus type is complex, and the adenovirus types that have been reported to cause urethritis are mainly HAdV-4 (E subgroup), HAdV-35 (B subgroup), HAdV-9, 37, 49, 56 (D subgroup). Adenovirus type 55 is a new virus produced by the recombination of human adenovirus types 11 and 14, and belongs to the B2 subgroup of group B. After homology sequence comparison, avoid areas with high homology when designing primers. The present application selects different coding genes of different pathogens to design primers, successfully avoiding interference and cross-talk between pathogens. The PGP coding gene is selected for Chlamydia trachomatis (CT), the UreB coding gene is selected for Ureaplasma urealyticum (UU), the UreA coding gene is selected for Ureaplasma parvum (UP), the GAP coding gene is selected for Mycoplasma hominis (MH), the MGP coding gene is selected for Mycoplasma genitalium (MG), the PorA coding gene is selected for Neisseria gonorrhoeae (NG), the US5 coding gene is selected for herpes simplex virus type I (HSV-I), the US3 coding gene is selected for herpes simplex virus type II (HSV-II), the P1 coding gene is selected for adenovirus (ADV), the proteinD coding gene is selected for Haemophilus influenzae (HI), and the ribosomalRNA coding gene is selected for Trichomonas vaginalis (TV).

[0012] In response to defect 2), the present application designs multiple pairs of primers for each pathogen. After repeated screening of multiple pairs of primers for each pathogen, the present application ultimately provides a primer and probe combination for detecting 11 NGU pathogens, which has high and consistent detection efficiency, accurate test results, and can quickly complete pathogen diagnosis at low cost.

[0013] In response to defect 3), after screening out the primer pairs with the best specificity and the closest amplification efficiency for each pathogen, the present application further utilizes the preference of the PCR instrument to balance the PCR amplification efficiency and fluorescence value of each pathogen, and further balances the amplification efficiency of each pathogen by increasing the amount of primers for pathogens with slightly lower detection efficiency. Finally, the detection limit of the 11 pathogens in the kit of the present application reached 2 copies / μL, with high sensitivity and balance.

[0014] Specifically, this application provides the following technical solutions:

[0015] In the first aspect of the present application, the present application proposes a primer set, which is used to detect urogenital tract pathogens, wherein the urogenital tract pathogens include: Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Ureaplasma parvum (UP), Mycoplasma hominis (MH), adenovirus (ADV), Trichomonas vaginalis (TV), herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), Haemophilus influenzae (HI) and Neisseria gonorrhoeae (NG).

[0016] In some examples of the present application, the aforementioned primer set may also include at least one of the following additional technical features:

[0017] In some examples of the present application, the aforementioned primer set sequences are shown in SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32 (Table 1).

[0018] In some preferred examples of the present application, the primer sequences for detecting Ureaplasma urealyticum are shown as SEQ ID NO: 1-2.

[0019] In some preferred examples of the present application, the primer sequences for detecting Chlamydia trachomatis are shown as SEQ ID NO: 4-5.

[0020] In some preferred examples of the present application, the primer sequences for detecting Mycoplasma genitalium are shown as SEQ ID NOs: 7-8.

[0021] In some preferred examples of the present application, the primer sequences for detecting Ureaplasma parvum are shown as SEQ ID NO: 10-11.

[0022] In some preferred examples of the present application, the primer sequences for detecting Mycoplasma hominis are shown as SEQ ID NOs: 13-14.

[0023] In some preferred examples of the present application, the primer sequences for detecting adenovirus are shown as SEQ ID NOs: 16-17.

[0024] In some preferred examples of the present application, the primer sequences for detecting Trichomonas vaginalis are shown as SEQ ID NOs: 19-20.

[0025] In some preferred examples of the present application, the primer sequences for detecting herpes simplex virus I are shown as SEQ ID NOs: 22-23.

[0026] In some preferred examples of the present application, the primer sequences for detecting herpes simplex virus II are shown as SEQ ID NOs: 25-26.

[0027] In some preferred examples of the present application, the primer sequences for detecting Haemophilus influenzae are shown as SEQ ID NOs: 28-29.

[0028] In some preferred examples of the present application, the primer sequences for detecting Neisseria gonorrhoeae are shown as SEQ ID NOs: 31-32.

[0029] Based on the above primer set, this application pioneered the use of urine specimens for non-gonococcal urethritis (NGU) pathogen detection, and the test results are highly consistent with those of traditional secretion specimens. The pathogen detection samples of this application support self-sampling at home, which improves patient compliance and reduces the frequency of visits and cross-infection risks.

[0030] The multiplex test based on the aforementioned primer set covers more than 99% of the pathogen types that can cause NGU. It has high specificity and sensitivity, can quickly determine the type of infectious pathogen, and guide clinical precision medication, thereby shortening the treatment cycle and improving cure efficiency. At the same time, it reduces the workload of doctors and reduces the risk of pathogen transmission.

[0031] In the second aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the aforementioned kit includes: any primer set of the first aspect.

[0032] In some examples of the present application, the aforementioned kit may also include at least one of the following additional technical features:

[0033] In some examples of the present application, the aforementioned kit further includes: a fluorescent probe for detecting urogenital tract pathogens. The probe used in the present application is a TaqMan probe labeled with FAM at the 5' end, and oligonucleotides labeled with a fluorescent reporter group (R) and a fluorescent quencher group (Q) at both ends of the probe. When the probe is intact, that is, in a random state and a state without PCR product hybridization, the fluorescence emitted by the reporter group is absorbed by the quencher group. During the fluorescent PCR amplification process, when the specific PCR product undergoes hybridization reaction with the TaqMan probe, the 5' end exonuclease activity of the HotStart Taq enzyme also cleaves the probe, and the fluorescence released by the reporter group can be detected by the fluorometer built into the PCR detector. The fluorescent reporter groups of these 11 probes can be selected from FAM, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, ROX, TexasRed, LCRED640 and LCRED705, etc.; the quenching group can be selected from MGB, BHQ0, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse and NFQ, etc. The principle of selecting the quenching group is that the fluorescence absorption spectrum of the quenching group overlaps with the emission spectrum of the fluorescent reporter group.

[0034] In some examples of the present application, the aforementioned fluorescent probe sequence is shown in any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, and 33.

[0035] In some preferred examples of the present application, the probe sequence for detecting Ureaplasma urealyticum is shown as SEQ ID NO:3.

[0036] In some preferred examples of the present application, the probe sequence for detecting Chlamydia trachomatis is shown as SEQ ID NO:6.

[0037] In some preferred examples of the present application, the probe sequence for detecting Mycoplasma genitalium is shown as SEQ ID NO:9.

[0038] In some preferred examples of the present application, the probe sequence for detecting Ureaplasma pumila is shown as SEQ ID NO:12.

[0039] In some preferred examples of the present application, the probe sequence for detecting Mycoplasma hominis is shown as SEQ ID NO: 15.

[0040] In some preferred examples of the present application, the probe sequence for detecting adenovirus is shown as SEQ ID NO:18.

[0041] In some preferred examples of the present application, the probe sequence for detecting Trichomonas vaginalis is shown in SEQ ID NO: 21

[0042] In some preferred examples of the present application, the probe sequence for detecting herpes simplex virus type I is shown as SEQ ID NO:24.

[0043] In some preferred examples of the present application, the probe sequence for detecting herpes simplex virus type II is shown as SEQ ID NO:27.

[0044] In some preferred examples of the present application, the probe sequence for detecting Haemophilus influenzae is shown as SEQ ID NO:30.

[0045] In some preferred examples of the present application, the probe sequence for detecting Neisseria gonorrhoeae is shown as SEQ ID NO:33.

[0046] In some examples of the present application, the aforementioned kit further includes an internal reference primer and an internal reference fluorescent probe.

[0047] In some preferred examples of the present application, the aforementioned internal reference primer sequences are shown as SEQ ID NOs: 34-35.

[0048] In some preferred examples of the present application, the aforementioned internal reference fluorescent probe sequence is shown as SEQ ID NO:36.

[0049] In some examples of the present application, the aforementioned kit further includes: reaction reagents, positive controls, and negative controls.

[0050] In some examples of the present application, the aforementioned reaction reagents include: HotStart Taq enzyme, UNG enzyme, dATP, dUTP, dCTP, dGTP, MgCl2. The present application adopts a UNG enzyme / dUTP anti-contamination system, which can reduce the contamination interference caused by the previous PCR reaction product.

[0051] It is understandable that, in addition to the above reagents, the kit may also include some commonly used reagents, such as enzyme-free water, instructions, etc.

[0052] This kit is the first to use urine that can be collected at home without invasive means as a specimen for NGU pathogen DNA testing, avoiding the need for professionals to collect samples or invasive risks. The kit can efficiently and evenly detect almost all pathogens that can cause NGU, which can improve the diagnosis and treatment rate of NGU, reduce the spread of NGU infection pathogens, and reduce the adverse consequences of female infertility, infertility, intrauterine infection, miscarriage, and other adverse consequences caused by persistent or repeated NGU infection, as well as male gonad infection, abnormal semen biochemical composition and production of antisperm antibodies, sperm deformity, decreased number, decreased activity rate, decreased activity, slower sperm motility, changes in motility, and even infertility. The prevalence of NGU in infertile patients is 59.67%.

[0053] The kit of this application adopts Taqman probe fluorescent PCR technology, and can obtain test results within 3 hours for 11 NGU infection pathogens through one sampling and one detection, with strong specificity and sensitivity of up to 2 copies / μL. It provides support for accurate and timely treatment of NGU infection, especially can greatly shorten the cure time of mixed infection and non-major pathogen infection, reduce the trouble of patients going back and forth to the hospital for diagnosis and treatment, and reduce the workload of doctors, improve the efficiency of diagnosis and treatment, and reduce the cost of diagnosis and treatment.

[0054] The kit of the present application can detect known NGU infection pathogens at one time. After clarifying the NGU infection pathogens, it can avoid the irrational use of antibiotics, reduce the generation and spread of drug-resistant bacteria, and effectively alleviate the pressure of antibiotic resistance.

[0055] In the third aspect of the present application, the present application proposes the use of any primer set of the first aspect or any kit of the second aspect in detecting urogenital tract pathogens.

[0056] The aforementioned primer sets or kits have the advantages of high sensitivity and strong specificity in detecting urogenital tract pathogens, and are suitable for urine samples, and can achieve non-invasive sample collection without the need for professional technicians.

[0057] In the fourth aspect of the present application, the present application proposes a method for detecting urogenital tract pathogens. According to an embodiment of the present application, the aforementioned method comprises: using any primer set of the first aspect or any kit of the second aspect to amplify the sample to be tested; detecting the amplified product to determine the type of urogenital tract pathogen in the aforementioned sample to be tested; wherein the sample to be tested is selected from DNA in male urethral secretions, female vaginal secretions or urine.

[0058] In some examples of the present application, the aforementioned method may further include at least one of the following additional technical features:

[0059] In some preferred examples of the present application, the aforementioned sample to be tested is selected from DNA in urine.

[0060] In some examples of the present application, the aforementioned amplification is performed in an amplification reaction system, in which the final concentration of the primer is 100nM-1000nM.

[0061] In some examples of the present application, in the aforementioned amplification reaction system, the final concentration of the probe is 50nM-500nM.

[0062] In some examples of the present application, the reaction procedure of the aforementioned amplification reaction is shown in Table 1;

[0063] Table 1 Reaction procedure

[0064]

[0065] Those skilled in the art will appreciate that the reaction temperature, reaction time or number of cycles may be fine-tuned and optimized based on actual experimental requirements.

[0066] In some examples of the present application, the test results include a fluorescence curve and a Ct value. If the Ct value is not greater than a first predetermined threshold, it indicates that the sample to be tested is positive for urogenital pathogens; if the Ct value is not less than a second predetermined threshold, it indicates that the sample to be tested is negative for urogenital pathogens; if the Ct value is greater than the first predetermined threshold and less than the second predetermined threshold, the sample to be tested is retested.

[0067] In some examples of the present application, the aforementioned predetermined threshold value may be a cutoff value (cutoff value) obtained after a large number of sample tests. It will be appreciated by those skilled in the art that differences in sample size may result in changes in the cutoff value. In some examples of the present application, the aforementioned first predetermined threshold value is selected from 37, and the aforementioned second predetermined threshold value is selected from 40.

[0068] The primer probes used in this method are designed for the specific conserved region sequences of 11 known NGU infection pathogens, and have strong detection specificity; they can detect the target gene sequence at a concentration of 2 copies / μL, and have high detection sensitivity; they can simultaneously detect 11 known NGU infection pathogens, covering all currently recognized NGU infection pathogens, and have high detection throughput; they are suitable for non-invasive home self-collected urine specimens, and sampling is convenient and non-invasive; the detection process is simple and fast, and can be completed within 3 hours from specimen submission to result; the result interpretation is clear and objective, and the results can also be qualitatively analyzed; a single test can identify the NGU infection pathogen, which helps doctors and patients to accurately use drugs for pathogens, improve diagnosis and treatment efficiency, and reduce the pain, time and cost of patients during infection; the entire detection process does not contain toxic and harmful substances, does not require post-processing of PCR products, and is harmless to operators and the environment.

[0069] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0071] Figure 1 A schematic diagram of the PCR amplification curve of a urethral (vaginal) secretion sample provided in an embodiment of the present application;

[0072] Figure 2 A schematic diagram of the PCR amplification curve of a urine sample provided in an embodiment of the present application;

[0073] Figure 3 A schematic diagram of the sensitivity test results for detecting Ureaplasma microti provided in the embodiments of the present application;

[0074] Figure 4 A schematic diagram of the sensitivity test results for detecting Trichomonas vaginalis provided in an embodiment of the present application;

[0075] Figure 5A schematic diagram of the sensitivity test results for detecting Mycoplasma genitalium provided in an embodiment of the present application;

[0076] Figure 6 A schematic diagram of the sensitivity test results for detecting herpes simplex virus type I provided in an embodiment of the present application;

[0077] Figure 7 A schematic diagram of the sensitivity test results for detecting Chlamydia trachomatis provided in the embodiments of the present application;

[0078] Figure 8 A schematic diagram of the results of a sensitivity test for detecting Mycoplasma hominis provided in an embodiment of the present application;

[0079] Fig. 9 A schematic diagram of the results of a sensitivity test for detecting adenovirus provided in an embodiment of the present application;

[0080] Fig.10 A schematic diagram of the sensitivity test results for detecting Neisseria gonorrhoeae provided in the embodiments of the present application;

[0081] Fig.11 A schematic diagram of the sensitivity test results for detecting herpes simplex virus type II provided in an embodiment of the present application;

[0082] Fig.12 A schematic diagram of the sensitivity test results for detecting Ureaplasma urealyticum provided in an embodiment of the present application;

[0083] Fig.13 A schematic diagram of the sensitivity test results for detecting Haemophilus influenzae provided in an embodiment of the present application;

[0084] Fig.14 A schematic diagram of the specificity test results for detecting Ureaplasma microti, Trichomonas vaginalis and Mycoplasma genitalium provided in the embodiments of the present application;

[0085] Fig.15 A schematic diagram of specific test results for detecting herpes simplex virus type I, Chlamydia trachomatis, Mycoplasma hominis and adenovirus provided in the examples of the present application;

[0086] Fig.16 A schematic diagram of specific test results for detecting Neisseria gonorrhoeae, herpes simplex virus type II, Ureaplasma urealyticum and Haemophilus influenzae provided in the embodiments of the present application;

[0087] Fig.17 A schematic diagram of the specific test results for detecting Ureaplasma microti provided in the embodiments of the present application;

[0088] Fig.18 A schematic diagram of the specific test results for detecting Trichomonas vaginalis provided in the embodiments of the present application;

[0089] Fig.19 A schematic diagram of the specific test results for detecting Mycoplasma genitalium provided in the embodiments of the present application;

[0090] Fig. 20 A schematic diagram of specific test results for detecting herpes simplex virus type I provided in an embodiment of the present application;

[0091] Fig.21 A schematic diagram of the specific test results for detecting Chlamydia trachomatis provided in the embodiments of the present application;

[0092] Fig. 22 A schematic diagram of the specific test results for detecting Mycoplasma hominis provided in the embodiments of the present application;

[0093] Fig.23 A schematic diagram of specific test results for detecting adenovirus provided in an embodiment of the present application;

[0094] Fig.24 A schematic diagram of specific test results for detecting Neisseria gonorrhoeae provided in an embodiment of the present application;

[0095] Fig.25 A schematic diagram of specific test results for detecting herpes simplex virus type II provided in an embodiment of the present application;

[0096] Fig.26 A schematic diagram of specific test results for detecting Ureaplasma urealyticum provided in an embodiment of the present application;

[0097] Fig. 27 A schematic diagram of the specific test results for detecting Haemophilus influenzae provided in the embodiments of the present application;

[0098] Fig.28A A schematic diagram of the NGU routine inspection process provided in an embodiment of the present application;

[0099] Fig.28B A schematic diagram of the NGU multiplex inspection process provided in an embodiment of the present application. DETAILED DESCRIPTION

[0100] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0101] In this article, unless otherwise specified, the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the present application described here can be implemented in a sequence other than those illustrated or described here. In the present application, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method, system, product or server comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices. In the description of the present application, unless otherwise specified, "multiple" refers to two or more than two.

[0102] The sequences in this application are shown in Table 2.

[0103] Table 2

[0104]

[0105]

[0106] Note: The naming convention for the description part is: pathogen name / internal reference-forward primer / reverse primer / probe; for example, UU-F is the forward primer sequence for amplifying Ureaplasma urealyticum; UU-R is the reverse primer sequence for amplifying Ureaplasma urealyticum; UU-P is the fluorescent probe sequence for detecting Ureaplasma urealyticum; the naming conventions for other pathogens are the same and will not be repeated here due to space limitations.

[0107] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0108] Example 1: NGU pathogen detection process

[0109] This embodiment uses the kit of the technical solution of the present application to detect 11 NGU pathogens, and the kit includes the following components:

[0110] PCR buffer1 / 2 / 3: PCR buffer, dATP, dUTP, dCTP, dGTP and MgCl2, HotStart Taq enzyme and UNG enzyme, primer probe mixture;

[0111] Positive control: plasmid containing amplified gene sequences corresponding to the above 11 NGU pathogens and the internal reference gene GAPDH;

[0112] Negative control: RNase-free water.

[0113] In the PCR reaction system, the final concentration of the primer in the amplification system is 100nM-1000nM; the final concentration of the probe in the amplification system is 50nM-500nM. The sequences of the primers and probes are shown in Table 2, and the reaction system is shown in Table 3.

[0114] Table 3 Reaction system

[0115]

[0116]

[0117] Kit operation process

[0118] 1. Sample preparation: Add 5 μL of sample DNA / RNA (extracted from human urine, urethral (vaginal) secretion specimens), negative and positive controls into different PCR reaction tubes, and add 15 μL of PCR buffer 1 / 2 / 3 to each tube to form a system, cover the tube caps, and place them in a fluorescent quantitative PCR instrument for fluorescent PCR detection.

[0119] 2. Amplification: Set the conditions for the PCR amplification reaction in the instrument to: 37°C, 2 min; 95°C, 5 min; 95°C, 15 s; 60°C, 45 s; 45 cycles.

[0120] 3. Analysis: After the reaction is completed, the baseline is set to automatic adjustment, and the test results are analyzed according to the amplification curve and Ct value.

[0121] 4. Validity determination:

[0122] Control quality control: The Ct value detected by RNase-free water is Undet or ≥40, and the Ct value detected by the positive control is ≤32, otherwise the experiment is considered invalid;

[0123] Internal standard gene: In sample detection, the Ct value of the internal standard gene channel is ≤30, and the amplification curve has an obvious exponential growth period.

[0124] 5. Interpretation of results:

[0125] Target gene: If the Ct value of the sample detection well is Undet or ≥40, the sample result is judged to be negative, and the sample to be tested does not contain target gene DNA or the content is below the detection limit;

[0126] If the Ct value of the sample detection hole is ≤37, the sample result is judged to be positive for the corresponding pathogen and the sample detection is successful;

[0127] The Ct value of the sample detection well is 37-40, and it needs to be retested. If the retest Ct value is <40 and the amplification curve has an obvious exponential growth period, it is judged as positive; the rest are negative.

[0128] Figure 1 , 2 It is a PCR amplification curve diagram of the present invention, wherein the positive quality control product detects an amplification curve, and the Ct value is less than 32, and the negative control has no amplification curve, indicating that the result is valid; the Ct value of the amplification curve of the weak positive sample is 37-40, and it is recommended to re-test. If the Ct value of the re-test result is less than 40 and there is an S-shaped amplification curve, it is positive, otherwise it is negative; the medium and strong samples all have amplification curves, and the Ct value is less than 37, which is determined to be a positive result.

[0129] Example 2: Multiplex qPCR sensitivity testing

[0130] The specific sequences of 11 pathogens and the sequences of the internal reference genes were connected to the pUC57 vector to synthesize plasmid standards. The plasmid construction was completed by Sangon Biotech (Shanghai) Co., Ltd. These plasmids were diluted in a 10-fold gradient, with a total of six gradients of 2×10 4 , 2×10 3 , 2×10 2 , 2×10 1 , 2×10 0 , 2×10 -1 copies / μL.

[0131] The gradient 2×10 4 , 2×10 3 , 2×10 2 , 2×10 1 , 2×10 0 , 2×10 -1 The minimum plasmid concentration that can be detected by the MqPCR detection method was determined by testing the plasmid template with 100 copies / μL. The results are as follows Figure 3-13 As shown in the figure, the detection limit of 11 pathogens can reach 2 copies / μL.

[0132] Example 3: Multiplex qPCR specificity testing

[0133] Since multiplex fluorescence quantitative PCR requires the detection of 3 or 4 pathogens and 1 internal reference gene in one system, the concentration of 2×10 3 2.5 μL of copies / μL plasmid standard was mixed as template according to Table 4, and multiplex fluorescence quantitative PCR detection well specificity test was performed. The test results are shown in Figure 14-16The results showed that there was no cross-reaction between the pathogens in each group, indicating that the detection primers had good specificity in multiplex fluorescence quantitative PCR; the concentration of 2×10 3 5 μL of copies / μL plasmid standard was mixed as template according to Table 4, and the specificity test in the multiplex fluorescence quantitative PCR detection kit was performed. The test results are shown in Figure 17-27 The results showed that there was no interference reaction between the pathogens in each group, indicating that the detection primers had good specificity in multiplex fluorescence quantitative PCR.

[0134] Table 4 Template preparation

[0135]

[0136] Example 4: Clinical sample testing

[0137] 1. Sample Collection

[0138] 1.1 Urine sample: Before collecting the specimen, clean the urethra thoroughly, collect 40mL of the first urine in the morning or the first urine after urination for more than 2 hours into the collection cup; carefully place the urine in the collection cup into the urine sample collection tube, make the liquid level reach the 45mL mark (with a warning line), and tighten the bottle cap firmly; turn the collection tube upside down to ensure that there is no leakage, and then gently shake the collection tube to ensure that the sample and the preservation solution are fully mixed. DNA extraction can be performed immediately, or it can be stored at room temperature for no more than 7 days or at -20℃±4℃ for no more than 6 months, and the number of freeze-thaw times shall not exceed 5 times.

[0139] 1.2 Male urethral secretions: Do not urinate for at least 1 hour before sampling. Clean the urethral opening with sterile saline, wipe it dry with sterile gauze or cotton balls, use a male swab to collect purulent secretions overflowing from the urethral opening, or insert the male swab 2 to 4 cm into the urethra to collect secretions, and place the swab for PCR testing in the cell preservation solution, bend the middle of the sampler to break it and leave the front part in the preservation solution. Gently shake the collection tube to ensure that the sample and the preservation solution are fully mixed. If there is no pus overflow, massage from the ventral surface of the penis toward the glans to promote secretion overflow. DNA extraction can be performed immediately, or it can be stored at room temperature for no more than 7 days or at -20℃±4℃ for no more than 6 months, and the number of freeze-thaw cycles should not exceed 5 times.

[0140] 1.3 Female vaginal secretions: Clean your hands, put on gloves and take out the sampler; stand with legs apart or sit half-lying, hold the handle with one hand, hold the rotating head at the tail with the other hand, and point the brush head toward yourself; slowly push the sampler into the vagina until the safety baffle touches the vaginal opening and twist the rotating head clockwise 5 to 6 times; slowly move the sampler out of the vagina, align the sponge head with the preservation liquid tube mouth to break the sampler, soak the sponge brush head in the preservation liquid, tighten the tube cover, mix evenly, and the sample is the sample to be tested. DNA extraction can be performed immediately, or it can be stored at room temperature for no more than 7 days or -20℃±4℃ for no more than 6 months, and the number of freeze-thaw times should not exceed 5 times.

[0141] 2. Sample extraction

[0142] 2.1 Urine sample:

[0143] 1) Take out 10-15 mL of urine sample (frozen urine sample needs to be thawed at room temperature and fully shaken to mix) and put it into a new 15 mL centrifuge tube. Centrifuge at 5000 rpm for 10 min and discard the supernatant until about 2 mL of liquid remains.

[0144] 2) Mix the liquid and precipitate by pipetting. Add the suspension to a 2 mL centrifuge tube, centrifuge at 12000 rpm for 5 minutes, and discard the supernatant.

[0145] 3) Add 500 μL of lysis solution NA and 30 μL of proteinase K to the above centrifuge tube, mix by pipetting, incubate at 58°C for 30 min, add 300 μL of isopropanol and 30 μL of magnetic beads N, and mix on a rotator for 10 min (make sure the magnetic beads do not sink to the bottom).

[0146] 4) Place the centrifuge tube on the magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely adsorbed to the side wall of the centrifuge tube, carefully remove the liquid.

[0147] 5) Add 800 μL of NW1 rinse solution to a 2 mL centrifuge tube, shake and mix for 10 seconds, centrifuge for 2 to 4 seconds, place on a magnetic stand for 2 minutes, and slowly and gently discard the supernatant. Repeat the above steps to absorb all the liquid and avoid absorbing the magnetic beads.

[0148] 6) Add 800 μL of NW2 rinse solution to a 2 mL centrifuge tube, shake and mix for 10 seconds, centrifuge for 2 to 4 seconds, place on a magnetic stand for 2 minutes, and slowly and gently discard the supernatant. Repeat the above steps to aspirate all the liquid and avoid aspirating the magnetic beads.

[0149] 7) Add 40-100 μL of elution buffer TE to a 2 mL centrifuge tube, gently shake to mix, place in a 65°C water bath or metal bath for incubation for 5 min, then take out the centrifuge tube, centrifuge for 10 s, magnetically absorb for 2-5 min, transfer the supernatant to a clean 200 μL centrifuge tube to obtain the required DNA solution, and store at -20±5°C for future use. For long-term storage, please place at -70°C.

[0150] 2.2 Secretion samples:

[0151] 1) Vortex the swab sample, take out 1000 μL into a 2 mL centrifuge tube, centrifuge at 12000 rpm for 2 min, and discard the supernatant.

[0152] 2) Add 500 μL of lysis solution TSL and 20 μL of proteinase K to the above centrifuge tube, mix by pipetting, and incubate at 58°C for 30 min. After lysis, add 350 μL of isopropanol and 30 μL of magnetic beads T, and mix on a rotator for 10 min (make sure the magnetic beads do not sink to the bottom).

[0153] 3) Place the centrifuge tube on the magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely adsorbed to the side wall of the centrifuge tube, carefully remove the liquid.

[0154] 4) Add 800 μL of washing solution TW1 to a 2 mL centrifuge tube, shake and mix for 10 seconds, centrifuge for 2 to 4 seconds, place on a magnetic stand for 2 minutes, and slowly and gently discard the supernatant. Repeat the above steps to absorb all the liquid and avoid absorbing the magnetic beads.

[0155] 5) Add 800 μL of washing solution TW2 to a 2 mL centrifuge tube, shake and mix for 10 seconds, centrifuge for 2 to 4 seconds, place on a magnetic stand for 2 minutes, and slowly and gently discard the supernatant. Repeat the above steps to absorb all the liquid and avoid absorbing the magnetic beads.

[0156] 6) Add 40-100 μL of elution buffer TE to a 2 mL centrifuge tube, gently shake to mix, place in a 65°C water bath or metal bath for incubation for 5 min, then take out the centrifuge tube, centrifuge for 10 s, magnetically absorb for 2-5 min, transfer the supernatant to a new 1.5 ml centrifuge tube to obtain the required DNA solution, and store at -20±5°C for future use. For long-term storage, please place at -70°C.

[0157] 3. Sample testing

[0158] 1) Add 5 μL of DNA template, RNase-free water and positive control extracted from human urine and urethral (vaginal) secretion specimens to different PCR reaction tubes, and add 15 μL of PCR buffer 1 / 2 / 3 to each tube, respectively, to form a system, cover the tube caps, and put them into the fluorescence quantitative PCR instrument for fluorescence PCR detection.

[0159] 2) Set the conditions of the PCR amplification reaction in the instrument as follows: digestion at 37°C for 2 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s; annealing at 60°C for 45 s; and 45 cycles.

[0160] 3) After the reaction is completed, the baseline is set to automatic adjustment, and the test results are analyzed according to the amplification curve and Ct value.

[0161] 4) Validity determination:

[0162] Control quality control: The Ct value detected by RNase-free water is Undet or ≥40, and the Ct value detected by the positive control is ≤32, otherwise the experiment is considered invalid;

[0163] Internal standard gene: In sample detection, the Ct value of the internal standard gene channel is ≤30, and the amplification curve has an obvious exponential growth period.

[0164] 5) Interpretation of results:

[0165] Target gene: If the Ct value of the sample detection well is Undet or ≥40, the sample result is judged to be negative, and the sample to be tested does not contain target gene DNA or the content is below the detection limit;

[0166] If the Ct value of the sample detection hole is ≤37, the sample result is judged to be positive for the corresponding pathogen and the sample detection is successful;

[0167] The Ct value of the sample detection well is 37-40, and it needs to be retested. If the retest Ct value is <40 and the amplification curve has an obvious exponential growth period, it is judged as positive; the rest are negative.

[0168] 4. Test results

[0169] 99 clinical samples were collected and tested by qPCR method. The test results were recorded and shown in Table 5.

[0170] Table 5 NGU clinical sample test results

[0171]

[0172]

[0173]

[0174] 5. Results Analysis

[0175] 1) Among the 99 suspected NGU cases, 4 tested negative using this kit, 3 of them were asymptomatic and their spouses were tested for NGU, and 1 had symptoms but none of the 11 pathogens included in this kit were detected, so it is possible that the patient was infected with other pathogens.

[0176] 2) 99 suspected NGU cases were tested with this kit, and 95 positive cases were found. There were 48 cases infected or co-infected with pathogens other than UU, UP, CT, and MG that are not suitable for tetracycline treatment. 50.5% of the cases needed to adjust the treatment drugs after testing, otherwise the NGU infection could not be completely cured.

[0177] The symptoms of NGU caused by different pathogens are similar, but the treatment of different pathogens is very different. Tetracyclines are generally the first choice for CT, MG, and UU infections, and macrolides are the second choice. Because MH carries the tetracycline-resistant tetM gene in the genome, the use of tetracycline is limited, and MH is naturally resistant to 14- and 15-ring macrolide antibiotics. Currently, quinolones are mainly used to treat MH infections in clinical practice. Ceftriaxone is the first choice for NG treatment, and spectinomycin is the second choice. TV infections are generally treated with metronidazole. HSV and ADV infections are generally treated as viral infections, and quinolones and macrolides are the first choice for HI infections. What clinicians need more is a combination test that covers most of the pathogens of NGU, so as to determine the right medicine after the infection, especially when the proportion of mixed infections of NGU continues to increase, which can shorten the patient's recovery time.

[0178] Example 5: Non-invasive home urine sample test result compliance rate

[0179] In order to determine the accuracy of urine specimens and urethral (specimen) secretions in detecting NGU pathogens, urine and urethral (vaginal) secretion specimens from 99 clinical cases were collected and tested respectively using the qPCR method. The test results were recorded and shown in Table 6.

[0180] Table 6 Comparison of test results of urine and urethral (vaginal) secretion samples in NGU clinical cases

[0181]

[0182]

[0183]

[0184]

[0185] Results Analysis

[0186] The test results of urine specimens and urethral (vaginal) secretion specimens of 99 cases were completely consistent with each other in 94 cases, and incompletely consistent in 5 cases. Among them, one urine specimen detected one more pathogen, and the other 4 urine specimens detected one less pathogen. Taking the urethral (vaginal) secretion specimens as the benchmark, the detection compliance rate of urine secretions reached 96%. The results show that the use of the kit of this application to detect non-invasive home-collected urine specimens can accurately detect the pathogens infected by NGU patients, providing reliable support for the timely and accurate treatment of NGU patients. At the same time, with the high rate of mycoplasma resistance today, it provides a convenient specimen for further research and development of molecular detection kits for mycoplasma resistance in urogenital system infections.

[0187] Example 6: Comparison of routine inspection and inspection process of this kit

[0188] For a long time, the traditional culture and immunological methods for diagnosing the pathogens of nongonococcal urethritis (NGU) have had a certain degree of missed diagnosis or misdiagnosis. The diagnosis is made based on a comprehensive analysis of epidemiological history, clinical manifestations and laboratory tests. The diagnosis of NGU must first exclude NG infection. At present, patients who are considered to have NGU in clinical practice mainly test for the common pathogens of UU, CT, MG and MH. For patients whose test results of the above common pathogens are negative or turn negative after treatment, but whose symptoms persist, it is recommended to test for pathogens such as TV, HSV, and ADV. The treatment of NGU should follow the principles of timely, adequate and standardized medication; different treatment plans should be adopted according to different conditions and drug sensitivity test results; follow-up should be carried out after treatment; and sexual partners should be examined and treated at the same time.

[0189] For example, case number 15, in routine testing ( Fig.28A ), NG examination and treatment → common pathogens UU / CT / MG / MH examination and treatment → rare pathogens TV / HSV / ADV examination and treatment, multiple visits to the doctor and multiple tests are required, and the diagnosis and treatment requires that the last pathogen test result is negative or turns negative after treatment, but other pathogen tests are recommended for patients with persistent symptoms, which consumes time and money, increases the burden on doctors, and prolongs the pain of patients; currently, except for NG, CT, UU, and MG pathogens, which have molecular detection methods, the remaining pathogens are commonly tested by culture methods, which have long culture cycles, are easily contaminated by liquid cultures, and have false positives. NGU is a sexually transmitted disease, and there are patients who are reluctant to seek medical treatment for privacy reasons, which delays the best treatment time for patients and leads to the spread of pathogens.

[0190] In the present invention, the inspection process can be completed by sampling and testing once ( Fig.28B), multiple joint tests cover most of the pathogens related to NGU, and are used in clinical diagnosis to timely judge the condition and achieve early, rapid and accurate treatment of the disease. Using non-invasive and convenient home sampling methods, multiple joint tests can accurately use drugs after confirming the infected pathogens, shorten the patient's recovery time, reduce the workload of doctors, and reduce the spread of pathogens.

[0191] It should be noted that the method of detecting NGU pathogen DNA using urine specimens is the core of the present invention. Ordinary technicians in this field are inspired by this patent to change, modify, replace, increase and reduce the above-mentioned pathogen combination without departing from the principles and purpose of this application.

[0192] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0193] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in the field can change, modify, replace and modify the above embodiments within the scope of the present application without departing from the principles and purpose of the present application.

Claims

1. A primer set, characterized in that: For the detection of urogenital pathogens; Among them, the urogenital tract pathogens include: Ureaplasma urealyticum, Chlamydia trachomatis, Mycoplasma genitalium, Ureaplasma parvum, Mycoplasma hominis, adenovirus, Trichomonas vaginalis, herpes simplex virus type I, herpes simplex virus type II, Haemophilus influenzae and Neisseria gonorrhoeae.

2. The primer set according to claim 1, characterized in that The primer set sequences are shown in SEQ ID NOs: 1-2, 4-5, 7-8, 10-11, 13-14, 16-17, 19-20, 22-23, 25-26, 28-29, 31-32; Preferably, the primer sequences for detecting Ureaplasma urealyticum are shown in SEQ ID NO: 1-2; Preferably, the primer sequences for detecting Chlamydia trachomatis are shown in SEQ ID NO: 4-5; Preferably, the primer sequences for detecting Mycoplasma genitalium are as shown in SEQ ID NO: 7-8; Preferably, the primer sequences for detecting Ureaplasma microti are shown in SEQ ID NO: 10-11; Preferably, the primer sequences for detecting Mycoplasma hominis are shown in SEQ ID NO: 13-14; Preferably, the primer sequences for detecting adenovirus are shown in SEQ ID NO: 16-17; Preferably, the primer sequences for detecting Trichomonas vaginalis are shown in SEQ ID NOs: 19-20; Preferably, the primer sequences for detecting herpes simplex virus type I are shown in SEQ ID NOs: 22-23; Preferably, the primer sequences for detecting herpes simplex virus type II are as shown in SEQ ID NOs: 25-26; Preferably, the primer sequences for detecting Haemophilus influenzae are as shown in SEQ ID NOs: 28-29; Preferably, the primer sequences for detecting Neisseria gonorrhoeae are shown as SEQ ID NOs: 31-32.

3. A kit, characterized in that: include: The primer set according to claim 1 or 2.

4. The kit according to claim 3, characterized in that Further including: Fluorescent probes for the detection of urogenital pathogens; Optionally, the fluorescent probe sequence is shown in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33; Preferably, the probe sequence for detecting Ureaplasma urealyticum is as shown in SEQ ID NO: 3; Preferably, the probe sequence for detecting Chlamydia trachomatis is as shown in SEQ ID NO: 6; Preferably, the probe sequence for detecting Mycoplasma genitalium is as shown in SEQ ID NO: 9; Preferably, the probe sequence for detecting Ureaplasma microti is as shown in SEQ ID NO: 12; Preferably, the probe sequence for detecting Mycoplasma hominis is as shown in SEQ ID NO: 15; Preferably, the probe sequence for detecting adenovirus is as shown in SEQ ID NO: 18; Preferably, the probe sequence for detecting Trichomonas vaginalis is as shown in SEQ ID NO: 21; Preferably, the probe sequence for detecting herpes simplex virus I is as shown in SEQ ID NO: 24; Preferably, the probe sequence for detecting herpes simplex virus II is as shown in SEQ ID NO: 27; Preferably, the probe sequence for detecting Haemophilus influenzae is as shown in SEQ ID NO: 30; Preferably, the probe sequence for detecting Neisseria gonorrhoeae is as shown in SEQ ID NO: 33; Optionally, further comprising an internal reference primer and an internal reference fluorescent probe; Preferably, the internal reference primer sequence is shown in SEQ ID NO:34-35; Preferably, the internal reference fluorescent probe sequence is as shown in SEQ ID NO:

36.

5. The kit according to claim 4, characterized in that Further comprising: reaction reagents, positive controls and negative controls; Optionally, the reaction reagents include: HotStart Taq enzyme, UNG enzyme, dATP, dUTP, dCTP, dGTP, and MgCl2.

6. Use of the primer set according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 5 in detecting urogenital tract pathogens.

7. A method for detecting urogenital tract pathogens, characterized in that: include: Amplifying the sample to be tested using the primer set described in any one of claims 1 to 2 or the kit described in any one of claims 3 to 5; Detecting the amplified product to determine the type of urogenital tract pathogen in the sample to be tested; Wherein, the sample to be tested is selected from DNA in male urethral secretions, female vaginal secretions or urine.

8. The method according to claim 7, characterized in that The sample to be tested is selected from DNA in urine.

9. The method according to claim 8, characterized in that The amplification is carried out in an amplification reaction system, in which the final concentration of the primer is 100nM-1000nM; Optionally, in the amplification reaction system, the final concentration of the probe is 50nM-500nM; Optionally, the reaction procedure of the amplification reaction is as shown in Table 1; Table 1 Reaction procedure 10. The method according to any one of claims 7 to 9, characterized in that: The test results include fluorescence curve and Ct value; If the Ct value is not greater than the first predetermined threshold, it is an indication that the sample to be tested is positive for urogenital pathogens; If the Ct value is not less than a second predetermined threshold, it is an indication that the sample to be tested is negative for urogenital pathogens; If the Ct value is greater than the first predetermined threshold value and less than the second predetermined threshold value, the sample to be tested is retested.

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