A primer composition for detecting pathogens related to reproductive tract infection and their drug-resistant genes and its application

Abstract

The present invention relates to a primer composition for detecting pathogens related to reproductive tract infections and their drug-resistance genes, and its application. The detection targets of the primer composition include 69 pathogens related to reproductive tract infections, including HPV virus, candidiasis, trichomoniasis, sexually transmitted diseases, etc., as well as antibiotic-resistant sites strongly associated with Neisseria gonorrhoeae and Mycoplasma genitalium. The primers in the primer composition can be used individually for PCR amplification, and can also be used for multiplex PCR amplification in the same detection system. There is little mutual interference between primers, and there is no obvious non-specific amplification. In the case of up to 150 primers, a single-tube multiplex PCR amplification reaction can be efficiently achieved. A single test can achieve comprehensive screening of pathogens related to complex reproductive tract infections, as well as drug resistance detection of Neisseria gonorrhoeae and Mycoplasma genitalium, with the advantages of good specificity, high sensitivity, and high accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of high-throughput sequencing and gene detection, and relates to the detection of pathogens related to reproductive tract infections and their drug-resistant genes, and specifically to a primer composition and application thereof for detecting pathogens related to reproductive tract infections and their drug-resistant genes. Background Art

[0002] Reproductive tract infections (RTIs) are a group of infectious diseases caused by a variety of pathogenic microorganisms. These include human papillomavirus (HPV), which is associated with cervical cancer; candidiasis caused by fungi such as Candida albicans; and trichomoniasis caused by Trichomonas vaginalis. Sexually transmitted infections (STDs) also comprise a broad category, including bacteria such as Neisseria gonorrhoeae; herpes simplex virus type 1; and various STD-associated pathogens, including Treponema pallidum, Mycoplasma, Chlamydia, and Ureaplasma. Epidemiological surveys show that globally, over one million people acquire curable STDs every day, most of whom are asymptomatic. RTIs not only impact reproductive health and fertility but also quality of life. Therefore, the detection and identification of RT pathogens is crucial for early detection and treatment of STDs, reducing disease risk, and controlling infectious diseases.

[0003] Common detection methods for reproductive tract infections include smear microscopy, culture, colloidal gold, and nucleic acid detection, each with its own advantages and disadvantages. Smear microscopy and culture are simple to operate, but have low sensitivity and a limited detection range. Colloidal gold detection combines antigen-antibody immune reactions with colloidal gold labeling and tracing technology for qualitative detection, which is convenient, fast, and low-cost. However, due to its susceptibility to interference factors, its sensitivity and specificity are low. Nucleic acid detection is recommended as the preferred detection method due to its high sensitivity and specificity, and is particularly suitable for detecting asymptomatic infections.

[0004] Several documents have disclosed cases of using nucleic acid detection methods to detect reproductive tract pathogens. For example, Chinese patent CN112195277B discloses a primer probe set and kit for detecting human papillomavirus based on real-time fluorescence quantitative PCR, which can detect 18 high-risk HPV types; Chinese patent CN104651353B discloses a primer set and universal chip for detecting 22 HPV types; Chinese patent CN108624704B discloses a probe primer set for the combined detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium using fluorescence quantitative PCR, which can simultaneously detect three sexually transmitted pathogens; and Chinese patent CN112301169B discloses a primer combination and kit for detecting multiple pathogens related to reproductive tract infections based on a multiplex fluorescence quantitative PCR method, which can simultaneously detect 15 pathogens related to reproductive tract infections.

[0005] However, due to limitations in technical methodologies, the above technologies are unable to achieve comprehensive detection of HPV virus typing for complex reproductive tract infections, candidiasis, trichomoniasis, and sexually transmitted disease-related pathogens, and none of them include detection of drug resistance of sexually transmitted pathogens. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a primer composition and its application for detecting pathogens related to reproductive tract infections and their drug-resistant genes. The detection targets of the primer composition cover 69 pathogens related to reproductive tract infections, as well as drug-resistant genes for Neisseria gonorrhoeae and Mycoplasma genitalium. It can achieve comprehensive screening of complex pathogens related to reproductive tract infections in the same detection system and procedure, with good specificity, high sensitivity and high accuracy.

[0007] The first aspect of the present invention is to provide a primer composition for detecting pathogens related to reproductive tract infections and their drug resistance genes, the primer composition comprising at least one set of the following primers:

[0008] Group 1: primers for human papillomavirus type 6, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 1 and the reverse primer shown in SEQ ID NO: 2;

[0009] Group 2: primers for human papillomavirus type 11, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 3 and the reverse primer shown in SEQ ID NO: 4;

[0010] Group 3: primers for human papillomavirus type 34, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 5 and the reverse primer shown in SEQ ID NO: 6;

[0011] Group 4: primers for human papillomavirus type 40, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 7 and the reverse primer shown in SEQ ID NO: 8;

[0012] Group 5: primers for human papillomavirus type 42, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 9 and the reverse primer shown in SEQ ID NO: 10;

[0013] Group 6: primers for human papillomavirus type 43, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 11 and the reverse primer shown in SEQ ID NO: 12;

[0014] Group 7: primers for human papillomavirus type 44, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 13 and the reverse primer shown in SEQ ID NO: 14;

[0015] Group 8: primers for human papillomavirus type 54, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 15 and the reverse primer shown in SEQ ID NO: 16;

[0016] Group 9: primers for human papillomavirus type 57, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 17 and the reverse primer shown in SEQ ID NO: 18;

[0017] Group 10: primers for human papillomavirus type 61, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 19 and the reverse primer shown in SEQ ID NO: 20;

[0018] Group 11: primers for human papillomavirus type 67, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 21 and the reverse primer shown in SEQ ID NO: 22;

[0019] Group 12: primers for human papillomavirus type 69, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 23 and the reverse primer shown in SEQ ID NO: 24;

[0020] Group 13: primers for human papillomavirus type 70, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 25 and the reverse primer shown in SEQ ID NO: 26;

[0021] Group 14: primers for human papillomavirus type 71, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 27 and the reverse primer shown in SEQ ID NO: 28;

[0022] Group 15: primers for human papillomavirus type 72, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 29 and the reverse primer shown in SEQ ID NO: 30;

[0023] Group 16: primers for human papillomavirus type 81, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 31 and the reverse primer shown in SEQ ID NO: 32;

[0024] Group 17: primers for human papillomavirus type 83, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 33 and the reverse primer shown in SEQ ID NO: 34;

[0025] Group 18: primers for human papillomavirus type 84, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 35 and the reverse primer shown in SEQ ID NO: 36;

[0026] Group 19: primers for human papillomavirus type 89, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 37 and the reverse primer shown in SEQ ID NO: 38;

[0027] Group 20: primers for human papillomavirus type 16, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 39 and the reverse primer shown in SEQ ID NO: 40;

[0028] Group 21: primers for human papillomavirus type 18, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 41 and the reverse primer shown in SEQ ID NO: 42;

[0029] Group 22: primers for human papillomavirus type 31, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 43 and the reverse primer shown in SEQ ID NO: 44;

[0030] Group 23: primers for human papillomavirus type 33, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 45 and the reverse primer shown in SEQ ID NO: 46;

[0031] Group 24: primers for human papillomavirus type 35, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 47 and the reverse primer shown in SEQ ID NO: 48;

[0032] Group 25: primers for human papillomavirus type 39, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 49 and the reverse primer shown in SEQ ID NO: 50;

[0033] Group 26: primers for human papillomavirus type 45, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 51 and the reverse primer shown in SEQ ID NO: 52;

[0034] Group 27: primers for human papillomavirus type 51, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 53 and the reverse primer shown in SEQ ID NO: 54;

[0035] Group 28: primers for human papillomavirus type 52, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 55 and the reverse primer shown in SEQ ID NO: 56;

[0036] Group 29: primers for human papillomavirus type 56, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 57 and the reverse primer shown in SEQ ID NO: 58;

[0037] Group 30: primers for human papillomavirus type 58, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 59 and the reverse primer shown in SEQ ID NO: 60;

[0038] Group 31: primers for human papillomavirus type 59, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 61 and the reverse primer shown in SEQ ID NO: 62;

[0039] Group 32: primers for human papillomavirus type 66, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 63 and the reverse primer shown in SEQ ID NO: 64;

[0040] Group 33: primers for human papillomavirus type 68a, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 65 and the reverse primer shown in SEQ ID NO: 66;

[0041] Group 34: primers for human papillomavirus type 68b, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 67 and the reverse primer shown in SEQ ID NO: 68;

[0042] Group 35: primers for human papillomavirus type 26, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 69 and the reverse primer shown in SEQ ID NO: 70;

[0043] Group 36: primers for human papillomavirus type 53, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 71 and the reverse primer shown in SEQ ID NO: 72;

[0044] Group 37: primers for human papillomavirus type 73, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 73 and the reverse primer shown in SEQ ID NO: 74;

[0045] Group 38: primers for human papillomavirus type 82, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 75 and the reverse primer shown in SEQ ID NO: 76;

[0046] Group 39: primers for Trichomonas vaginalis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 77 and the reverse primer shown in SEQ ID NO: 78;

[0047] Group 40: primers for Lactobacillus iners, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 79 and the reverse primer shown in SEQ ID NO: 80;

[0048] Group 41: Primers for Lactobacillus crispatus, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 81 and the reverse primer shown in SEQ ID NO: 82:

[0049] Group 42: Primers targeting Atopobium vaginalis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 83 and the reverse primer shown in SEQ ID NO: 84:

[0050] Group 43: primers for Gardnerella vaginalis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 85 and the reverse primer shown in SEQ ID NO: 86;

[0051] Group 44: primers for Megasphaera type 1, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 87 and the reverse primer shown in SEQ ID NO: 88;

[0052] Group 45: primers targeting bacterial vaginosis-associated bacteria 2, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 89 and the reverse primer shown in SEQ ID NO: 90;

[0053] Group 46: primers for herpes simplex virus type 1, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 91 and the reverse primer shown in SEQ ID NO: 92;

[0054] Group 47: primers for herpes simplex virus type 2, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 93 and the reverse primer shown in SEQ ID NO: 94;

[0055] Group 48: primers against Neisseria gonorrhoeae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 95 and the reverse primer shown in SEQ ID NO: 96;

[0056] Group 49: primers for Haemophilus ducreyi, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 97 and the reverse primer shown in SEQ ID NO: 98;

[0057] Group 50: primers targeting Treponema pallidum, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 99 and the reverse primer shown in SEQ ID NO: 100;

[0058] Group 51: primers targeting Ureaplasma urealyticum, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 101 and the reverse primer shown in SEQ ID NO: 102;

[0059] Group 52: primers targeting Ureaplasma parvum, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 103 and the reverse primer shown in SEQ ID NO: 104;

[0060] Group 53: primers for Ureaplasma parvum type 1, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 105 and the reverse primer shown in SEQ ID NO: 106;

[0061] Group 54: primers against Ureaplasma parvum type 3, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 107 and the reverse primer shown in SEQ ID NO: 108;

[0062] Group 55: primers for Ureaplasma parvum type 6, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 109 and the reverse primer shown in SEQ ID NO: 110;

[0063] Group 56: primers against Ureaplasma parvum type 14, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 111 and the reverse primer shown in SEQ ID NO: 112;

[0064] Group 57: primers for Chlamydia trachomatis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 113 and the reverse primer shown in SEQ ID NO: 114;

[0065] Group 58: primers for Mycoplasma hominis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 115 and the reverse primer shown in SEQ ID NO: 116;

[0066] Group 59: primers for Mycoplasma genitalium, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 117 and the reverse primer shown in SEQ ID NO: 118;

[0067] Group 60: primers for Candida albicans, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 119 and the reverse primer shown in SEQ ID NO: 120;

[0068] Group 61: primers for Candida glabrata, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 121 and the reverse primer shown in SEQ ID NO: 122;

[0069] Group 62: primers for Pichia kudriavzevii, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 123 and the reverse primer shown in SEQ ID NO: 124;

[0070] Group 63: primers for Candida tropicalis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 125 and the reverse primer shown in SEQ ID NO: 126;

[0071] Group 64: primers for Candida parapsilosis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 127 and the reverse primer shown in SEQ ID NO: 128;

[0072] Group 65: primers for Candida dubliniensis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 129 and the reverse primer shown in SEQ ID NO: 130;

[0073] Group 66: primers for varicella-zoster virus, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 131 and the reverse primer shown in SEQ ID NO: 132;

[0074] Group 67: primers for cytomegalovirus, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 133 and the reverse primer shown in SEQ ID NO: 134;

[0075] Group 68: primers for Toxoplasma gondii, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 135 and the reverse primer shown in SEQ ID NO: 136;

[0076] Group 69: primers for Streptococcus agalactiae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 137 and the reverse primer shown in SEQ ID NO: 138;

[0077] Group 70: primers targeting the A2059G site of the 23S rRNA resistance gene of Neisseria gonorrhoeae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 139 and the reverse primer shown in SEQ ID NO: 140;

[0078] Group 71: primers targeting the 23S rRNA drug-resistance gene C2611T of Neisseria gonorrhoeae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 141 and the reverse primer shown in SEQ ID NO: 142;

[0079] Group 72: primers targeting the gyrA resistance gene of Neisseria gonorrhoeae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 143 and the reverse primer shown in SEQ ID NO: 144;

[0080] Group 73: primers targeting the Mycoplasma genitalium 23S rRNA resistance gene, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 145 and the reverse primer shown in SEQ ID NO: 146;

[0081] Group 74: Primers targeting the Mycoplasma genitalium parC resistance gene, whose nucleotide sequences are the forward primer shown in SEQ ID NO: 147 and the reverse primer shown in SEQ ID NO: 148.

[0082] In some embodiments, the primer composition includes groups 20-21.

[0083] In some embodiments, the primer composition further comprises at least one of the groups 22-38.

[0084] In some embodiments, the primer composition further comprises at least one of Group 39, Groups 48 to 51, Groups 57 to 60, and Groups 70 to 74.

[0085] In some embodiments, the primer composition further comprises at least one of Groups 40 to 45 and Group 69.

[0086] In some embodiments, the primer composition further comprises at least one of Groups 1 to 19, Groups 46 to 47, Groups 52 to 56, and Groups 61 to 68.

[0087] In some embodiments, the primer composition includes groups 1-74.

[0088] In some embodiments, the primer composition further comprises primers for an internal standard gene; preferably, the primers for the internal standard gene have nucleotide sequences such as the forward primer shown in SEQ ID NO: 149 and the reverse primer shown in SEQ ID NO: 150.

[0089] The second aspect of the present invention is to provide the use of the primer combination described above in the preparation of a product for detecting the reproductive tract infection-related pathogens and their drug-resistant genes.

[0090] In some embodiments, the product is a kit.

[0091] The third aspect of the present invention is to provide a kit for detecting pathogens related to reproductive tract infections and their drug-resistant genes, wherein the kit comprises the primer combination described above.

[0092] The fourth aspect of the present invention is to provide a method for detecting pathogens related to reproductive tract infections and their drug-resistant genes for non-diagnostic purposes, the method comprising the following steps: obtaining nucleic acid from the sample to be tested, performing PCR amplification using the primer composition as described above or the kit as described above, constructing a library, and sequencing.

[0093] The inventors of the present invention have obtained a primer combination for detecting pathogens related to reproductive tract infections and their drug resistance genes based on their many years of experience and research screening. The detection targets of the primer combination cover 69 pathogens related to reproductive tract infections, as well as antibiotic-related resistance sites strongly associated with Neisseria gonorrhoeae and Mycoplasma genitalium. The primers in the primer combination can be used alone for PCR amplification, and can also be used for multiple PCR amplification in the same detection system; the optimal amplification conditions of the primers are similar, and there is little mutual interference between the primers, ensuring that each amplicon can be successfully amplified under the same amplification conditions, without obvious non-specific amplification. With up to 150 primers, a single tube multiple PCR amplification reaction can be efficiently achieved, and a single test can achieve comprehensive screening of pathogens related to complex reproductive tract infections, as well as drug resistance detection of Neisseria gonorrhoeae and Mycoplasma genitalium, with the advantages of good specificity, high sensitivity and high accuracy. In addition, the primer combination is suitable for the detection of various sample types, including urine, vaginal secretions, cervical exfoliated cells, semen or prostatic fluid.

[0094] The detection method of the present invention, through the combination of multiplex PCR amplification and targeted high-throughput sequencing, can accurately detect the target pathogen. The entire process, from nucleic acid extraction to test result issuance, takes approximately 12 hours. It is particularly suitable for people with non-acute genital tract infections or those undergoing routine physical examinations. Furthermore, due to its wide detection range, it is also suitable for those seeking the cause of recurrent unexplained genital tract infections, effectively avoiding the problem of multiple tests and multiple sampling.

[0095] The primer composition and method provided by the present invention are of great significance for early detection and early treatment of clinical sexually transmitted diseases, reducing disease risks and controlling infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 This is the peak diagram of the library constructed in Example 2.

[0097] Figure 2 This is the peak diagram of the first generation sequencing in Example 3. DETAILED DESCRIPTION

[0098] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0099] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0100] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0101] Furthermore, as used herein, the term "or" is inclusive and equivalent to the term "and / or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0102] The present invention is further described in detail below with reference to specific embodiments.

[0103] Example 1

[0104] This example illustrates the detection target, primer sequence and detection method of the present invention.

[0105] 1. Pathogen targets: A total of 69 pathogen targets, as shown in Table 1 below:

[0106] Table 1 Combination of pathogens of reproductive tract infection

[0107]

[0108]

[0109] 2. Drug-resistant genes: At the same time, the detection scope includes genomic loci that are strongly associated with antibiotic resistance in Mycoplasma genitalium and Neisseria gonorrhoeae, as shown in Table 2 below:

[0110] Table 2 Drug resistance detection range

[0111]

[0112] 3. Primer Sequences: A large number of amplification primers were designed for the selected targets and drug resistance sites. These primers were screened to obtain a primer combination that closely matched the optimal amplification conditions, ensuring successful amplification of each amplicon under the same amplification conditions. The primers in this primer combination exhibited minimal mutual interference, resulting in excellent detection performance across all primers in a single system. The sequence information for the screened primer combination is shown in Table 3 below:

[0113] Table 3 Primer sequence information

[0114]

[0115]

[0116]

[0117]

[0118] 4. The 5' end of each primer is connected to a common sequence required for nucleic acid sequencing library construction. This common sequence is complementary to the 3' end of the sequencing adapter of the sequencing platform and serves to connect the sequencing adapter primers during amplification. Sequencing adapter primers can be conventional sequencing adapter primers for the corresponding sequencing platform. To match this, a sequence complementary to the common sequence is provided at the 3' end of each sequencing adapter primer to connect the amplified products of the target region. The following common sequence is preferably used in the present invention: 5'-GACTGCCGCTGGTTGGATG-3' (SEQ ID NO: 187).

[0119] 5. Specific primers and amplification enzymes are formulated to form a reagent system 1, and the extracted DNA nucleic acid is subjected to super-multiplex PCR amplification of the target region. The mixing ratio of the primers is shown in the pooling coefficient of each primer in Table 3 above. The primers are diluted to a specific concentration according to the pooling coefficient and mixed for subsequent amplification. The specific primer concentration is calculated as follows: Cn = Pn / Psum x 100, where n represents the primer number, which is an integer between 1 and 150 as shown in Table 3, Cn represents the concentration of the nth primer, in μM, Pn represents the pooling coefficient of the nth primer, and Psum represents the sum of the pooling coefficients of all primers.

[0120] 6. Target region fragments are magnetically purified and then amplified using sequencing adapter primers. The sequencing adapter primers and amplification enzyme are combined to form Library Amplification Reagent System 2. Library amplification is performed, resulting in a library structure of "sequencing adapter-common sequence-target region-common sequence-sequencing adapter."

[0121] 7. Each super-multiplex PCR library is purified by magnetic beads, and after purification, the libraries are mixed to obtain a library for detection of pathogenic microorganisms related to reproductive tract infections.

[0122] 8. The library is subjected to high-throughput sequencing, and the reads of the sequencing are aligned to the self-built genome database using a bioinformatics analysis method, the number of reads of the internal standard and each pathogen alignment is obtained, and quality control is performed. The nucleic acid sequencing platform includes the Ion torrent platform, the Roche454 platform, the SoLID sequencing platform, and the KM MiniSeqDx-CN sequencing platform. The software for aligning the reads of the sequencing to the self-built genome database includes Bowtie2, Minimap2, HISAT2, and BWA alignment software. The present invention preferably uses the KM MiniSeqDx-CN platform to perform high-throughput sequencing on the library, and uses the mem alignment algorithm in the BWA software to perform sequence alignment to obtain a BAM format file of the alignment result.

[0123] 9. For samples that pass quality control, we first perform bioinformatics statistical analysis to obtain the number of reads that map to pathogens. We then normalize these reads and compare the normalized value with a pre-set threshold to determine whether the pathogen detection result is negative or positive. The formula for normalizing reads is as follows: assuming the number of raw reads from a sample is raw_reads_num, and the number of reads identified as falling within the pathogen 1 target region after alignment is x, then the normalized read count x' = x * 100,000 / raw_reads_num. Normalization eliminates variations in the amount of raw data from each sample.

[0124] 10. For the detection of drug-resistant sites, if the pathogen detection result is determined to be positive, the bioinformatics software samtools is used to perform site depth and base statistics on the aligned BAM result file, obtain the number of reads of the reference base and mutant base of the target site, and calculate the mutation ratio. The size of the mutation ratio value is compared with the pre-set threshold to determine whether the pathogen resistance result is negative or positive.

[0125] Example 2 Clinical sample detection

[0126] The collected clinical samples were tested using the primer combination of the present invention in the following manner:

[0127] 1. Sample Collection: In the hospital's gynecology and men's outpatient departments, samples that had undergone molecular testing in the past month were retrospectively searched to ensure that these positive samples covered as many pathogens as possible within the scope of the present invention system. A total of 55 female vaginal secretion samples, 50 female cervical exfoliated cell samples, and 83 male urine samples were collected, for a total of 188 samples.

[0128] 2. Sample pretreatment: Place the sampling tube on a vortex mixer and vortex thoroughly for 30 seconds. Directly draw 1.3 mL of sample into a 1.5 mL sterile centrifuge tube as the sample to be tested.

[0129] 3. Nucleic acid extraction: Use nucleic acid extraction or purification reagents (Guangzhou Jinqirui Biotechnology Co., Ltd., registration number Yuesui Xiebei 20220884) according to the instructions for extraction.

[0130] 4. Preparation of primer mixture: Prepare all primers into primer mixture according to the ratio in Table 4 below. Prepare primer mixture according to the number of detection reactions, and each reaction requires 1.5 μL of primer mixture.

[0131] Taking the preparation of a 300 μL primer mixture as an example, the specific method is: after diluting the primers to 100 μM, according to the coefficients in Table 4, use a pipette to aspirate 2.2 μL of PRTI-1F and PRTI-1R, respectively, then use a pipette to aspirate 1.2 μL of PRTI-2F and PRTI-2R, respectively, and so on, until finally use a pipette to aspirate 2.1 μL of PRTI-75F and PRTI-75R, respectively. Mix thoroughly. The prepared primer mixture should be stored at 4 degrees Celsius and used within one week. For long-term storage, it should be placed at -20 degrees Celsius.

[0132] Table 4

[0133]

[0134]

[0135] 5. Library Preparation

[0136] (1) PCR1 reaction: Perform super-multiplex PCR amplification on the nucleic acid template. Prepare a 25 μL library amplification reaction system with 1.5 μL of multiple primer mix + 12.5 μL of PCR reaction buffer + 11 μL of nucleic acid to obtain the target region fragment. The amplification program is shown in Table 5 below:

[0137] Table 5

[0138]

[0139]

[0140] (2) After the PCR1 reaction product was purified, a 25 μL library amplification reaction system was prepared by combining 1 μL of adapter primers, 12.5 μL of PCR reaction buffer, and 11.5 μL of the purified enriched product. The library was amplified to obtain a library structure of "sequencing adapter-common sequence-target region-common sequence-sequencing adapter". The amplification procedure is shown in Table 6 below:

[0141] Table 6

[0142]

[0143] (3) Purify each super-multiplex PCR library, and after purification, mix the sample libraries to obtain a library for detection of reproductive tract-related pathogenic microorganisms. The library peak diagram is as follows Figure 1 shown.

[0144] 6. Sequencing: The sequencer used for sequencing was KMMiniSeqDx-CN produced by Guangzhou Jinqirui Biotechnology Co., Ltd., and the universal sequencing reaction kit (Guangzhou Jinqirui Biotechnology Co., Ltd., product number: KS107-CXR, specification MR100) was used. The library was sequenced according to the instructions.

[0145] 7. Analysis of Sequencing Data

[0146] The sequencing data obtained by testing the sample using the primer combination of the present invention can be analyzed according to the following method:

[0147] (1) Bioinformatics analysis: For the fastQ files obtained by sequencing, first use fastp software to count the amount of raw data, remove the adapter sequences, and filter the low-quality data in the original sequencing results. Then write a script to screen the valid reads with primer sequences in the sequencing products and retain them. Then use the mem alignment algorithm of the bwa alignment software to align the valid read product sequences to the self-built database, and count the number of reads covered by each pathogen based on the BED file. Among them, the above-mentioned self-built database is the genome matched according to the product sequence of the primer, which comes from the Nucleotide database of NCBI. Each pathogen target contains at least one matching genome. For the same target, try to add multiple genomes so that all possible product sequences of the primer can be perfectly aligned to the database. The above-mentioned BED file format is a text file format widely used in bioinformatics. It is specifically used to describe features and regions on the genome. It is generated based on the genomic region position information of the primer theoretical alignment to the self-built database.

[0148] (2) Sample quality control: Quality control is performed on the data obtained from the analysis. Preferably, the minimum sequencing data requirement for each sample is ≥50k, otherwise the original data is judged to be unqualified; the sequencing quality requirement Q30 ≥75%, otherwise the sequencing quality is judged to be unqualified; the internal standard normalized reads number is ≥50, otherwise it is judged to be unqualified. For the original number of reads assigned to each pathogen within the detection range, the normalized read number of the pathogen is calculated, and the pathogen is judged to be negative or positive based on the size of the normalized read number. Preferably, the normalized read number of the pathogen is ≥10, and it is judged to be positive, otherwise it is judged to be negative. The formula for normalizing the number of reads is: assuming that the original number of reads sequenced for a certain sample is raw_reads_num, and the number of reads confirmed to fall into the target area of ​​pathogen 1 after comparison is x, then the normalized read number x'=x*100000 / raw_reads_num. After normalization, the differences caused by the different amounts of original data for each sample can be eliminated.

[0149] (3) Quality control of drug resistance detection: When the pathogen detection result is determined to be positive, the bioinformatics software samtools is used to perform site depth and base statistics on the aligned BAM result file, obtain the number of reads of the reference base and the mutant base of the target site, and calculate the mutation ratio. The pathogen resistance result is determined to be negative or positive based on the size of the mutation ratio value and the pre-set threshold. Preferably, if the original number of reads covering the drug resistance site to be tested, that is, the depth value ≥30, it is determined to be a successful test, otherwise it is determined to be insufficient depth; under the premise of successful detection, if the number of reads of the mutant base accounts for, that is, the mutation ratio ≥10%, it is determined to be a drug resistance mutation, otherwise it is determined to be an undetectable drug resistance mutation.

[0150] According to the above quality control requirements, 188 samples in this example were analyzed and all passed the quality control. The statistical results of the test are shown in Table 7 below.

[0151] H-7

[0152]

[0153]

[0154] The results show that the primer combination of the present invention can be used to detect a variety of reproductive tract sample types with a high detection success rate. The positive rate of pathogen detection in retrospective clinical samples is high, and for Neisseria gonorrhoeae and Mycoplasma genitalium, drug-resistant site results can be normally detected.

[0155] The specific test results of each sample are shown in Table 8:

[0156] Table 8

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167] The statistical results of relevant pathogen detection are shown in Table 9:

[0168] Table 9

[0169]

[0170]

[0171] The above results show that the primers for different pathogens in the primer combination of the present invention can achieve simultaneous detection of multiple reproductive tract pathogens under the same system and procedure. Compared with the prior results, the detection method of the present invention can detect all the pathogens in the prior results, and in some samples, it can also detect additional pathogens with weak positive results that were not detected in the prior results. These pathogen detection sequences are all specific when compared to the database, indicating that the test results are accurate. This shows that the primer combination of the present invention has higher detection sensitivity and lower detection limit, and can achieve comprehensive and accurate detection of multiple reproductive tract pathogens in a single reaction system and procedure. It is very suitable for detection in people with non-acute reproductive tract infections, people with routine physical examinations, and people with repeated unexplained reproductive tract infections.

[0172] Example 3 Verification of drug resistance site accuracy

[0173] Based on Example 2, this example performed first-generation sequencing verification on clinical samples that tested positive for drug resistance in Neisseria gonorrhoeae and Mycoplasma genitalium. First-generation primer sequences were designed based on the sequences before and after the drug resistance sites, as shown in Table 10 below:

[0174] Table 10

[0175]

[0176] The above primers were used to perform first generation sequencing on the two drug-resistant positive samples in Example 2. The results are as follows: Figure 2 The system detection results are summarized in Table 11 below. The results show that the detection results of the primer combination of the present invention for drug-resistant sites in clinical samples are completely consistent with the first-generation sequencing results, indicating that the primer combination can successfully and correctly detect drug-resistant sites.

[0177] Table 11

[0178]

[0179]

[0180] Example 4 Detection Performance Test of the Primer Composition of the Present Invention

[0181] In this example, the detection performance of the primer composition of the present invention was tested using the detection of Streptococcus agalactiae, Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 39, and human papillomavirus type 51 as examples.

[0182] Among them, Streptococcus agalactiae was purchased from Qinsituo Biological, Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae were purchased from Guangzhou Bondeson Biological, and national standard strains of human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 39, and human papillomavirus type 51 were used.

[0183] The mixed negative urine samples of the above 8 pathogens to be tested were used as artificial simulated samples, and the following experimental design was carried out:

[0184] 1. Serial dilution: Nucleic acid extraction was performed according to the protocol in Example 2, and the extracted simulated sample nucleic acid was serially diluted to obtain detection concentrations of 250 copies / mL, 500 copies / mL, and 1000 copies / mL, respectively.

[0185] 2. Set up replicates: For each pathogen simulated sample, perform 20 replicates at three concentration gradients, then test the above samples according to the protocol in Example 1, obtaining a total of 480 library experimental results;

[0186] 3. According to the analysis scheme in Example 2, the normalized sequence number results obtained for the eight pathogens were statistically summarized; when calculating the detection rate, a pathogen with a normalized read number ≥ 10 was considered positive, otherwise it was considered negative. The results are shown in Table 12 below: Table 12

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] As can be seen from the results in the above table, the primer set of the present invention was used to detect samples of 8 positive pathogens with an accuracy rate of 100%. From the results of nucleic acid dilution and 20 repetitions, it can be seen that the detection rate of Streptococcus agalactiae, Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae was 100% (20 / 20) at a concentration of 500 copies / mL, while the detection rate of human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 39, and human papillomavirus type 51 was 100% (20 / 20) at a concentration of 1000 copies / mL.

[0193] Example 5 System Adjustment Performance Study

[0194] The present invention designs multiple pairs of candidate primers for each pathogen within the target detection range, and conducts a large amount of research, and finally obtains the primer combination that is close to the optimal amplification conditions of the present invention, with little mutual interference between primers and no obvious non-specific amplification, ensuring that each amplicon can be successfully amplified under the same amplification conditions. During the experiment, the system is adjusted according to the detection of dimers and the proportion of positive pathogens in the system. Specifically, first, the primer combination with an average of more than 8% of the dimer ratio detected in the system is replaced, and the top-ranked primer dimers are preferentially replaced so that the overall dimer ratio of the system is not higher than 25%. Secondly, the primers that miss positive pathogens or have low detection performance in the priori results are replaced, and finally the primer coefficient of the internal reference is adjusted so that different sample types can be successfully detected.

[0195] The detection results of the primer system (first round system) in Table 13 are used as an example for comparison. In addition to using the pathogen detection primers in Table 13 to replace the corresponding pathogen primers in Table 4 (final system) of the present invention, the primer compositions in the first round system are the same as those in Table 4.

[0196] Table 13

[0197]

[0198]

[0199] The comparison of the results of testing the two primer combination systems on 15 different types of clinical samples is shown in Table 14.

[0200] Table 14

[0201]

[0202]

[0203] The results showed that in the first round of the system, the average proportion of overall dimers was 53%, and the average maximum proportion of a single dimer combination was 16%, which did not meet the detection requirements. The normalized sequence number of the internal standard was low, and there were missed pathogens. The detection results of the final system of the present invention showed that the average proportion of overall dimers was 15%, and the average maximum proportion of a single dimer combination was 6%. The internal standards of all sample types passed the test, and compared with the prior results, no pathogens were missed, indicating that the primer system of the present invention has better detection performance.

[0204] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A primer composition for detecting pathogens related to reproductive tract infection and their drug resistance genes, characterized in that: The primer composition The following primer sets are included: Group 1: primers for human papillomavirus type 6, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 1 and the reverse primer shown in SEQ ID NO: 2; Group 2: primers for human papillomavirus type 11, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 3 and the reverse primer shown in SEQ ID NO: 4; Group 3: primers for human papillomavirus type 34, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 5 and the reverse primer shown in SEQ ID NO: 6; Group 4: primers for human papillomavirus type 40, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 7 and the reverse primer shown in SEQ ID NO: 8; Group 5: primers for human papillomavirus type 42, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 9 and the reverse primer shown in SEQ ID NO: 10; Group 6: primers for human papillomavirus type 43, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 11 and the reverse primer shown in SEQ ID NO: 12; Group 7: primers for human papillomavirus type 44, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 13 and the reverse primer shown in SEQ ID NO: 14; Group 8: primers for human papillomavirus type 54, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 15 and the reverse primer shown in SEQ ID NO: 16; Group 9: primers for human papillomavirus type 57, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 17 and the reverse primer shown in SEQ ID NO: 18; Group 10: primers for human papillomavirus type 61, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 19 and the reverse primer shown in SEQ ID NO: 20; Group 11: primers for human papillomavirus type 67, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 21 and the reverse primer shown in SEQ ID NO: 22; Group 12: primers for human papillomavirus type 69, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 23 and the reverse primer shown in SEQ ID NO: 24; Group 13: primers for human papillomavirus type 70, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 25 and the reverse primer shown in SEQ ID NO: 26; Group 14: primers for human papillomavirus type 71, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 27 and the reverse primer shown in SEQ ID NO: 28; Group 15: primers for human papillomavirus type 72, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 29 and the reverse primer shown in SEQ ID NO: 30; Group 16: primers for human papillomavirus type 81, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 31 and the reverse primer shown in SEQ ID NO: 32; Group 17: primers for human papillomavirus type 83, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 33 and the reverse primer shown in SEQ ID NO: 34; Group 18: primers for human papillomavirus type 84, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 35 and the reverse primer shown in SEQ ID NO: 36; Group 19: primers for human papillomavirus type 89, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 37 and the reverse primer shown in SEQ ID NO: 38; Group 20: primers for human papillomavirus type 16, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 39 and the reverse primer shown in SEQ ID NO: 40; Group 21: primers for human papillomavirus type 18, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 41 and the reverse primer shown in SEQ ID NO: 42; Group 22: primers for human papillomavirus type 31, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 43 and the reverse primer shown in SEQ ID NO: 44; Group 23: primers for human papillomavirus type 33, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 45 and the reverse primer shown in SEQ ID NO: 46; Group 24: primers for human papillomavirus type 35, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 47 and the reverse primer shown in SEQ ID NO: 48; Group 25: primers for human papillomavirus type 39, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 49 and the reverse primer shown in SEQ ID NO: 50; Group 26: primers for human papillomavirus type 45, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 51 and the reverse primer shown in SEQ ID NO: 52; Group 27: primers for human papillomavirus type 51, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 53 and the reverse primer shown in SEQ ID NO: 54; Group 28: primers for human papillomavirus type 52, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 55 and the reverse primer shown in SEQ ID NO: 56; Group 29: primers for human papillomavirus type 56, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 57 and the reverse primer shown in SEQ ID NO: 58; Group 30: primers for human papillomavirus type 58, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 59 and the reverse primer shown in SEQ ID NO: 60; Group 31: primers for human papillomavirus type 59, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 61 and the reverse primer shown in SEQ ID NO: 62; Group 32: primers for human papillomavirus type 66, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 63 and the reverse primer shown in SEQ ID NO: 64; Group 33: primers for human papillomavirus type 68a, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 65 and the reverse primer shown in SEQ ID NO: 66; Group 34: primers for human papillomavirus type 68b, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 67 and the reverse primer shown in SEQ ID NO: 68; Group 35: primers for human papillomavirus type 26, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 69 and the reverse primer shown in SEQ ID NO: 70; Group 36: primers for human papillomavirus type 53, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 71 and the reverse primer shown in SEQ ID NO: 72; Group 37: primers for human papillomavirus type 73, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 73 and the reverse primer shown in SEQ ID NO: 74; Group 38: primers for human papillomavirus type 82, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 75 and the reverse primer shown in SEQ ID NO: 76; Group 39: primers for Trichomonas vaginalis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 77 and the reverse primer shown in SEQ ID NO: 78; Group 40: primers for Lactobacillus iners, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 79 and the reverse primer shown in SEQ ID NO: 80; Group 41: primers for Lactobacillus crispatus, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 81 and the reverse primer shown in SEQ ID NO: 82; Group 42: primers targeting Atopobium vaginalis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 83 and the reverse primer shown in SEQ ID NO: 84; Group 43: primers targeting Gardnerella vaginalis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 85 and the reverse primer shown in SEQ ID NO: 86; Group 44: primers for Megasphaera type 1, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 87 and the reverse primer shown in SEQ ID NO: 88; Group 45: primers targeting bacterial vaginosis-associated bacteria 2, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 89 and the reverse primer shown in SEQ ID NO: 90; Group 46: primers for herpes simplex virus type 1, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 91 and the reverse primer shown in SEQ ID NO: 92; Group 47: primers for herpes simplex virus type 2, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 93 and the reverse primer shown in SEQ ID NO: 94; Group 48: primers for Neisseria gonorrhoeae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 95 and the reverse primer shown in SEQ ID NO: 96; Group 49: primers for Haemophilus ducreyi, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 97 and the reverse primer shown in SEQ ID NO: 98; Group 50: primers for Treponema pallidum, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 99 and the reverse primer shown in SEQ ID NO: 100; Group 51: primers for Ureaplasma urealyticum, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 101 and the reverse primer shown in SEQ ID NO: 102; Group 52: primers for Ureaplasma parvum, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 103 and the reverse primer shown in SEQ ID NO: 104; Group 53: primers for Ureaplasma parvum type 1, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 105 and the reverse primer shown in SEQ ID NO: 106; Group 54: primers against Ureaplasma parvum type 3, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 107 and the reverse primer shown in SEQ ID NO: 108; Group 55: primers for Ureaplasma parvum type 6, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 109 and the reverse primer shown in SEQ ID NO: 110; Group 56: primers for Ureaplasma parvum type 14, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 111 and the reverse primer shown in SEQ ID NO: 112; Group 57: primers for Chlamydia trachomatis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 113 and the reverse primer shown in SEQ ID NO: 114; Group 58: primers for Mycoplasma hominis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 115 and the reverse primer shown in SEQ ID NO: 116; Group 59: primers for Mycoplasma genitalium, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 117 and the reverse primer shown in SEQ ID NO: 118; Group 60: primers for Candida albicans, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 119 and the reverse primer shown in SEQ ID NO: 120; Group 61: primers for Candida glabrata, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 121 and the reverse primer shown in SEQ ID NO: 122; Group 62: primers for Pichia kudriavzevii, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 123 and the reverse primer shown in SEQ ID NO: 124; Group 63: primers for Candida tropicalis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 125 and the reverse primer shown in SEQ ID NO: 126; Group 64: primers for Candida parapsilosis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 127 and the reverse primer shown in SEQ ID NO: 128; Group 65: primers for Candida dubliniensis, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 129 and the reverse primer shown in SEQ ID NO: 130; Group 66: primers for varicella-zoster virus, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 131 and the reverse primer shown in SEQ ID NO: 132; Group 67: primers for cytomegalovirus, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 133 and the reverse primer shown in SEQ ID NO: 134; Group 68: primers for Toxoplasma gondii, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 135 and the reverse primer shown in SEQ ID NO: 136; Group 69: primers for Streptococcus agalactiae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 137 and the reverse primer shown in SEQ ID NO: 138; Group 70: primers targeting the A2059G site of the 23S rRNA resistance gene of Neisseria gonorrhoeae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 139 and the reverse primer shown in SEQ ID NO: 140; Group 71: primers targeting the 23S rRNA drug-resistance gene C2611T of Neisseria gonorrhoeae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 141 and the reverse primer shown in SEQ ID NO: 142; Group 72: primers targeting the gyrA resistance gene of Neisseria gonorrhoeae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 143 and the reverse primer shown in SEQ ID NO: 144; Group 73: primers targeting the Mycoplasma genitalium 23S rRNA resistance gene, the nucleotide sequences of which are the forward primer shown in SEQ ID NO: 145 and the reverse primer shown in SEQ ID NO: 146; Group 74: Primers targeting the Mycoplasma genitalium parC resistance gene, whose nucleotide sequences are the forward primer shown in SEQ ID NO: 147 and the reverse primer shown in SEQ ID NO:

148.

2. Use of the primer combination according to claim 1 in preparing a product for detecting pathogens related to reproductive tract infections and their drug-resistant genes.

3. A kit for detecting pathogens related to reproductive tract infection and their drug-resistant genes, characterized in that: The kit comprises the primer combination according to claim 1.

4. A method for detecting pathogens related to reproductive tract infection and their drug resistance genes for non-diagnostic purposes, characterized in that: The following steps are involved: The nucleic acid of the sample to be tested is obtained, and PCR amplification is performed using the primer combination as described in claim 1 or the kit as described in claim 3, a library is constructed, and sequencing is performed.

Images