Primer group and kit for high-throughput sequencing of targeted genital tract infection pathogens and application of primer group and kit

Abstract

The invention discloses a primer group and a kit for high-throughput sequencing of targeted genital tract infection pathogens and application of the primer group and the kit, microorganisms and related drug-resistant genes in a detection range are identified by combining multiple PCR (polymerase chain reaction) with high-throughput sequencing, and the sensitivity is high. The detection technology is applied to identification of genital tract infection pathogens and has important clinical significance, the operation steps are simplified, the detection cost is reduced, and the detection efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer set and a kit for high-throughput sequencing of pathogens targeting reproductive tract infections, and applications thereof. Background Art

[0002] Female lower genital tract infection is a common gynecological disease, with vaginitis and cervicitis being the most common, including but not limited to: bacterial vaginosis, aerobic vaginitis, vulvovaginal candidiasis, vaginal trichomoniasis, and cervicitis caused by Neisseria gonorrhoeae, Chlamydia, Mycoplasma, etc. Lower genital tract infection is closely related to a variety of gynecological and obstetric diseases, and can lead to infertility, female genital tract cancer, etc.

[0003] Microorganisms associated with female lower reproductive tract infections include: bacteria, fungi, viruses and special pathogens. Currently, the common detection methods in clinical practice are: culture method, morphological detection method, immunological detection method represented by colloidal gold / enzyme-linked immunosorbent assay and common nucleic acid detection method. Among them, the culture method mainly focuses on bacteria, and has weak detection capabilities for other types of pathogens, because a large number of pathogens are not easy to survive in an in vitro artificial culture environment; the morphological detection method is mainly based on the typical biological morphology of common pathogens for identification. Although the detection is rapid, the accuracy and stability are poor; the immunological detection method is convenient and fast, with low dependence on professional instruments and personnel, but the accuracy is low, and the false negative problem is prominent. In contrast, nucleic acid detection has better taken into account the cost, detection speed and detection performance, and has become the mainstream detection method. However, traditional nucleic acid detection technology has gradually failed to meet the clinical detection needs for complex infections, exposing serious shortcomings, including but not limited to: the types and numbers of detected pathogens are small, and it is unable to cope with complex clinical infections. It is easy to miss detection; it is difficult to detect multiple pathogens and corresponding drug-resistant genes / mutations at the same time, and it is not easy to provide coherent diagnosis and medication guidance; In order to better meet the needs of clinical testing, high-throughput sequencing technology targeting pathogens of reproductive tract infections has emerged. Pathogen targeted sequencing (tNGS) is a new generation of sequencing technology based on super-multiplex PCR amplification (or targeted capture) and high-throughput sequencing. It does not rely on traditional microbial culture. It directly enriches dozens to hundreds of known pathogenic microorganisms and their virulence and / or drug resistance genes in clinical samples, and then performs high-throughput sequencing. Then, it compares and analyzes with the database, and judges the types of pathogenic microorganisms contained in the sample based on the compared sequence information. It can quickly and objectively detect more pathogenic microorganisms in clinical samples to provide a basis for clinical diagnosis. It is particularly advantageous for the detection of low-concentration pathogens, especially the detection of their virulence and / or drug resistance genes.

[0004] Compared with the traditional detection methods mentioned above, it has many advantages and features: ⑴ It can detect hundreds of different types of pathogens. ⑵ The detection speed is fast, from receiving the sample to the final export of the formal test report, it is generally less than 24 hours. ⑶ The cost converted to a single pathogen is low. ⑷ It can detect a large number of pathogens and drug-resistant genes at the same time, providing more consistent diagnosis and medication guidance.

[0005] However, current high-throughput sequencing technologies targeting pathogens mainly focus on human respiratory tract-related pathogen infections, and lack detection technologies for reproductive tract pathogens, especially primers suitable for comprehensive screening of reproductive tract pathogens. Summary of the invention

[0006] In order to overcome the shortcomings of the existing high-throughput sequencing technology targeting pathogens, which has poor detection capabilities for reproductive tract pathogens, the present invention provides a primer set, a kit and its application for high-throughput sequencing of pathogens targeting reproductive tract infections. The present invention is implemented by the following technical solutions: A primer set for high-throughput sequencing of pathogens of reproductive tract infections, comprising: HPV16 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 253, and the rear primer sequence is shown in seq ID No. 254; HPV18 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 255, and the rear primer sequence is shown in seq ID No. 256; HPV26 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 257, and the rear primer sequence is shown in seq ID No. 258; HPV31 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 259, and the rear primer sequence is shown in seq ID No. 260; HPV33 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 261, and the rear primer sequence is shown in seq ID No. 262; HPV35 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 263, and the rear primer sequence is shown in seq ID No. 264; HPV39 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 265, and the rear primer sequence is shown in seq ID No. 266; HPV45 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 267, and the rear primer sequence is shown in seq ID No. 268; HPV51 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 269, and the rear primer sequence is shown in seq ID No. 2700; HPV52 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 271, and the rear primer sequence is shown in seq ID No. 272; HPV53 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 273, and the rear primer sequence is shown in seq ID No. 274; HPV56 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 275, and the rear primer sequence is shown in seq ID No. 276; HPV58 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 277, and the rear primer sequence is shown in seq ID No. 278; HPV59 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 279, and the rear primer sequence is shown in seq ID No. 280; HPV66 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 281, and the rear primer sequence is shown in seq ID No. 282; HPV67 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 283, and the rear primer sequence is shown in seq ID No. 284; HPV68 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 285, and the rear primer sequence is shown in seq ID No. 286; HPV73 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 287, and the rear primer sequence is shown in seq ID No. 288; HPV82 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.289, and the rear primer sequence is shown in seq ID No.290.

[0007] Optionally, at least one of the following detection primers is also included: HPV6 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 291, and the rear primer sequence is shown in seq ID No. 292; HPV11 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 293, and the rear primer sequence is shown in seq ID No. 294; HPV40 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 295, and the rear primer sequence is shown in seq ID No. 296; HPV42 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 297, and the rear primer sequence is shown in seq ID No. 298; HPV43 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 299, and the rear primer sequence is shown in seq ID No. 300; HPV54 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 301, and the rear primer sequence is shown in seq ID No. 302; HPV61 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 303, and the rear primer sequence is shown in seq ID No. 304; HPV70 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.305, and the rear primer sequence is shown in seq ID No.306; HPV72 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 307, and the rear primer sequence is shown in seq ID No. 308; HPV81 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 309, and the rear primer sequence is shown in seq ID No. 310; HPV89 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.311, and the rear primer sequence is shown in seq ID No.312; HPV34 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.313, and the rear primer sequence is shown in seq ID No.314; HPV44 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.315, and the rear primer sequence is shown in seq ID No.316; HPV57 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.317, and the rear primer sequence is shown in seq ID No.318; HPV69 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.319, and the rear primer sequence is shown in seq ID No.320; HPV71 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 321, and the rear primer sequence is shown in seq ID No. 322; HPV83 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 323, and the rear primer sequence is shown in seq ID No. 324; HPV84 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.325, and the rear primer sequence is shown in seq ID No.326.

[0008] Optionally, it also includes at least one of primers for detecting beneficial symbiotic bacteria, primers for detecting anaerobic / facultative anaerobic bacteria, primers for detecting aerobic bacteria, primers for detecting STDs bacteria, primers for detecting mycobacteria, primers for detecting high-risk / suspected high-risk human papillomavirus, primers for detecting low-risk human papillomavirus, primers for detecting other DNA viruses, primers for detecting RNA viruses, primers for detecting fungi, and primers for detecting parasites; and / or The beneficial symbiotic bacteria detection primers include: Primers for detecting Lactobacillus crispatus, the sequence of the front primer is shown in seq ID No.1, and the sequence of the rear primer is shown in seq ID No.2; Primers for detecting Lactobacillus iners, the forward primer sequence is shown in seq ID No.3, and the rear primer sequence is shown in seq ID No.4; The primers for detecting Lactobacillus jensenii, the sequence of the front primer is shown in seq ID No.5, and the sequence of the rear primer is shown in seq ID No.6; Lactobacillus gasseri detection primers, the front primer sequence is shown in seq ID No.7, and the rear primer sequence is shown in seq ID No.8; Lactobacillus detection primers, the forward primer sequence is shown in seq ID No.9, and the rear primer sequence is shown in seq ID No.10; The anaerobic / facultative anaerobic bacteria detection primers include: Primers for detecting Gardnerella vaginalis, the forward primer sequence is shown in seq ID No. 11, and the rear primer sequence is shown in seq ID No. 12; The primers for detecting Atopobium vaginalis, the forward primer sequence is shown in seq ID No. 13, and the rear primer sequence is shown in seq ID No. 14; Primers for detecting Bacteroides fragilis, the forward primer sequence is shown in seq ID No. 19, and the rear primer sequence is shown in seq ID No. 20; Two-way Prevotella detection primers, the forward primer sequence is shown in seq ID No. 21, and the rear primer sequence is shown in seq ID No. 22; Proteus mirabilis detection primers, the forward primer sequence is shown in seq ID No. 23, and the rear primer sequence is shown in seq ID No. 24; Streptococcus agalactiae detection primers, the front primer sequence is shown in seq ID No. 25, and the rear primer sequence is shown in seq ID No. 26; Primers for detecting Streptococcus pneumoniae, the forward primer sequence is shown in seq ID No. 27, and the rear primer sequence is shown in seq ID No. 28; Anaerobic peptostreptococcus detection primers, the forward primer sequence is shown in seq ID No. 29, and the rear primer sequence is shown in seq ID No. 30; Primers for detecting Fingoldella magna, the forward primer sequence is shown in seq ID No. 31, and the rear primer sequence is shown in seq ID No. 32; Micromonas microti detection primers, the forward primer sequence is shown in seq ID No. 33, and the rear primer sequence is shown in seq ID No. 34; Primers for detecting Clostridium perfringens, the forward primer sequence is shown in seq ID No.35, and the rear primer sequence is shown in seq ID No.36; Mobiluncus timidus detection primers, the forward primer sequence is shown in seq ID No. 37, and the rear primer sequence is shown in seq ID No. 38; Mobiluncus cruzi detection primers, the forward primer sequence is shown in seq ID No.39, and the rear primer sequence is shown in seq ID No.40; The primers for detecting giant cocci, the sequence of the front primer is shown in seq ID No.41, and the sequence of the rear primer is shown in seq ID No.42; Actinomyces ildrichthyes detection primers, the forward primer sequence is shown in seq ID No.43, and the rear primer sequence is shown in seq ID No.44; Oral trichomoniasis detection primers, the forward primer sequence is shown in seq ID No.45, and the rear primer sequence is shown in seq ID No.46; The primers for detecting Megasphaera elsdenii, the sequence of the front primer is shown in seq ID No.47, and the sequence of the rear primer is shown in seq ID No.48; Primers for aerobic bacteria detection include: Staphylococcus aureus detection primers, the forward primer sequence is shown in seq ID No.49, and the rear primer sequence is shown in seq ID No.50; Primers for detecting Staphylococcus epidermidis, the sequence of the front primer is shown in seq ID No.51, and the sequence of the rear primer is shown in seq ID No.52; Primers for detecting Staphylococcus intermedius, the forward primer sequence is shown in seq ID No.53, and the rear primer sequence is shown in seq ID No.54; Primers for detecting hemolytic Staphylococcus aureus, the forward primer sequence is shown in seq ID No.55, and the rear primer sequence is shown in seq ID No.56; Primers for detecting Staphylococcus lugdunensis, the forward primer sequence is shown in seq ID No.57, and the rear primer sequence is shown in seq ID No.58; Enterococcus faecalis detection primers, the forward primer sequence is shown in seq ID No.59, and the rear primer sequence is shown in seq ID No.60; Enterococcus faecium detection primers, the forward primer sequence is shown in seq ID No.61, and the rear primer sequence is shown in seq ID No.62; Escherichia coli detection primers, the forward primer sequence is shown in seq ID No.63, and the rear primer sequence is shown in seq ID No.64; Klebsiella pneumoniae detection primers, the forward primer sequence is shown in seq ID No.65, and the rear primer sequence is shown in seq ID No.66; Primers for detecting Klebsiella oxytoca, the forward primer sequence is shown in seq ID No.67, and the rear primer sequence is shown in seq ID No.68; The primers for detecting Klebsiella aerogenes, the sequence of the front primer is shown in seq ID No.69, and the sequence of the rear primer is shown in seq ID No.70; Enterobacter cloacae complex detection primers, the forward primer sequence is shown in seq ID No.71, and the rear primer sequence is shown in seq ID No.72; Primers for detecting Citrobacter cohnii, the forward primer sequence is shown in seq ID No.73, and the rear primer sequence is shown in seq ID No.74; Primers for detecting Citrobacter freundii, the forward primer sequence is shown in seq ID No.75, and the rear primer sequence is shown in seq ID No.76; Pseudomonas aeruginosa detection primers, the forward primer sequence is shown in seq ID No.79, and the rear primer sequence is shown in seq ID No.80; Haemophilus influenzae detection primers, the forward primer sequence is shown in seq ID No.81, and the rear primer sequence is shown in seq ID No.82; Haemophilus parainfluenzae detection primers, the forward primer sequence is shown in seq ID No.83, and the rear primer sequence is shown in seq ID No.84; Primers for detecting Streptococcus anginosus, the sequence of the front primer is shown in seq ID No.85, and the sequence of the rear primer is shown in seq ID No.86; STDs bacterial detection primers include: The primers for detecting Neisseria gonorrhoeae, the sequence of the front primer is shown in seq ID No.87, and the sequence of the rear primer is shown in seq ID No.88; The primers for detecting Treponema pallidum, the sequence of the front primer is shown in seq ID No.89, and the sequence of the rear primer is shown in seq ID No.90; Haemophilus ducreyi detection primers, the forward primer sequence is shown in seq ID No.91, and the rear primer sequence is shown in seq ID No.92; Chlamydia trachomatis detection primers, the forward primer sequence is shown in seq ID No.93, and the rear primer sequence is shown in seq ID No.94; Mycoplasma hominis detection primers, the forward primer sequence is shown in seq ID No.95, and the rear primer sequence is shown in seq ID No.96; Ureaplasma urealyticum detection primers, the front primer sequence is shown in seq ID No.97, and the rear primer sequence is shown in seq ID No.98; Mycoplasma genitalium detection primers, the front primer sequence is shown in seq ID No.99, and the rear primer sequence is shown in seq ID No.100; The mycobacterium detection primers include: Mycobacterium tuberculosis complex detection primers, the front primer sequence is shown in seq ID No. 103, and the rear primer sequence is shown in seq ID No. 104; High-risk and suspected high-risk human papillomavirus detection primers: HPV16 detection primers, the forward primer sequence is shown in seq ID No. 135, and the rear primer sequence is shown in seq ID No. 136; HPV18 detection primers, the forward primer sequence is shown in seq ID No. 137, and the rear primer sequence is shown in seq ID No. 138; HPV31 detection primers, the forward primer sequence is shown in seq ID No. 139, and the rear primer sequence is shown in seq ID No. 140; HPV33 detection primers, the forward primer sequence is shown in seq ID No. 141, and the rear primer sequence is shown in seq ID No. 142; HPV35 detection primers, the forward primer sequence is shown in seq ID No. 143, and the rear primer sequence is shown in seq ID No. 144; HPV39 detection primers, the forward primer sequence is shown in seq ID No. 145, and the rear primer sequence is shown in seq ID No. 146; HPV45 detection primers, the forward primer sequence is shown in seq ID No. 147, and the rear primer sequence is shown in seq ID No. 148; HPV51 detection primers, the forward primer sequence is shown in seq ID No. 149, and the rear primer sequence is shown in seq ID No. 150; HPV52 detection primers, the forward primer sequence is shown in seq ID No. 151, and the rear primer sequence is shown in seq ID No. 152; HPV56 detection primers, the forward primer sequence is shown in seq ID No. 153, and the rear primer sequence is shown in seq ID No. 154; HPV58 detection primers, the forward primer sequence is shown in seq ID No. 155, and the rear primer sequence is shown in seq ID No. 156; HPV59 detection primers, the forward primer sequence is shown in seq ID No. 157, and the rear primer sequence is shown in seq ID No. 158; HPV66 detection primers, the forward primer sequence is shown in seq ID No. 159, and the rear primer sequence is shown in seq ID No. 160; HPV68 detection primers, the forward primer sequence is shown in seq ID No. 161, and the rear primer sequence is shown in seq ID No. 162; HPV26 detection primers, the forward primer sequence is shown in seq ID No. 163, and the rear primer sequence is shown in seq ID No. 164; HPV53 detection primers, the forward primer sequence is shown in seq ID No. 165, and the rear primer sequence is shown in seq ID No. 166; HPV67 detection primers, the forward primer sequence is shown in seq ID No. 167, and the rear primer sequence is shown in seq ID No. 168; HPV70 detection primers, the forward primer sequence is shown in seq ID No. 169, and the rear primer sequence is shown in seq ID No. 170; HPV73 detection primers, the forward primer sequence is shown in seq ID No. 171, and the rear primer sequence is shown in seq ID No. 172; HPV82 detection primers, the forward primer sequence is shown in seq ID No. 173, and the rear primer sequence is shown in seq ID No. 174; Low-risk human papillomavirus detection primers include: HPV6 detection primers, the forward primer sequence is shown in seq ID No. 175, and the rear primer sequence is shown in seq ID No. 176; HPV11 detection primers, the forward primer sequence is shown in seq ID No. 177, and the rear primer sequence is shown in seq ID No. 178; HPV40 detection primers, the forward primer sequence is shown in seq ID No. 179, and the rear primer sequence is shown in seq ID No. 180; HPV42 detection primers, the forward primer sequence is shown in seq ID No. 181, and the rear primer sequence is shown in seq ID No. 182; HPV43 detection primers, the forward primer sequence is shown in seq ID No. 183, and the rear primer sequence is shown in seq ID No. 184; HPV54 detection primers, the forward primer sequence is shown in seq ID No. 185, and the rear primer sequence is shown in seq ID No. 186; HPV61 detection primers, the forward primer sequence is shown in seq ID No. 187, and the rear primer sequence is shown in seq ID No. 188; HPV72 detection primers, the forward primer sequence is shown in seq ID No. 189, and the rear primer sequence is shown in seq ID No. 190; HPV81 detection primers, the forward primer sequence is shown in seq ID No. 191, and the rear primer sequence is shown in seq ID No. 192; HPV89 detection primers, the forward primer sequence is shown in seq ID No. 193, and the rear primer sequence is shown in seq ID No. 194; Other DNA virus detection primers include: Human herpes virus type I detection primers, the front primer sequence is shown in seq ID No.209, and the rear primer sequence is shown in seqID No.210; Human herpes virus type II detection primers, the front primer sequence is shown in seq ID No.211, and the rear primer sequence is shown in seq ID No.212; Epstein-Barr virus detection primers, the forward primer sequence is shown in seq ID No. 213, and the rear primer sequence is shown in seq ID No. 214; Cytomegalovirus detection primers, the forward primer sequence is shown in seq ID No. 215, and the rear primer sequence is shown in seq ID No. 216; Primers for detecting molluscum contagiosum virus, the sequence of the front primer is shown in seq ID No. 217, and the sequence of the rear primer is shown in seq ID No. 218; Hepatitis B virus detection primers, the front primer sequence is shown in seq ID No. 219, and the rear primer sequence is shown in seq ID No. 220; Human polyomavirus type 1 detection primers, the forward primer sequence is shown in seq ID No. 221, and the rear primer sequence is shown in seq ID No. 222; Human polyomavirus type 2 detection primers, the forward primer sequence is shown in seq ID No. 223, and the rear primer sequence is shown in seq ID No. 224; Varicella-zoster virus detection primers, the forward primer sequence is shown in seq ID No. 225, and the rear primer sequence is shown in seq ID No. 226; RNA virus detection primers include: Human immunodeficiency virus type I detection primers, the forward primer sequence is shown in seq ID No.125, and the rear primer sequence is shown in seq ID No.126; Human immunodeficiency virus type II detection primers, the forward primer sequence is shown in seq ID No.127, and the rear primer sequence is shown in seq ID No.128; Hepatitis A virus detection primers, the front primer sequence is shown in seq ID No.129, and the rear primer sequence is shown in seq ID No.130; Hepatitis C virus detection primers, the front primer sequence is shown in seq ID No.131, and the rear primer sequence is shown in seq ID No.132; Human T-lymphotropic virus type 1 detection primers, the forward primer sequence is shown in seq ID No.133, and the rear primer sequence is shown in seq ID No.134; Fungal detection primers include: Candida albicans detection primers, the forward primer sequence is shown in seq ID No. 105, and the rear primer sequence is shown in seq ID No. 106; Primers for detecting Candida tropicalis, the sequence of the front primer is shown in seq ID No. 107, and the sequence of the rear primer is shown in seq ID No. 108; Primers for detecting Candida glabrata, the sequence of the front primer is shown in seq ID No. 109, and the sequence of the rear primer is shown in seq ID No. 110; Candida krusei detection primers, the forward primer sequence is shown in seq ID No.111, and the rear primer sequence is shown in seq ID No.112; Primers for detecting Portuguese Candida albicans, the sequence of the front primer is shown in seq ID No. 113, and the sequence of the rear primer is shown in seq ID No. 114; Primers for detecting Candida parapsilosis, the sequence of the front primer is shown in seq ID No.115, and the sequence of the rear primer is shown in seq ID No.116; Primers for detecting Candida dulio, the forward primer sequence is shown in seq ID No.117, and the rear primer sequence is shown in seq ID No.118; Parasite detection primers include: Trichomonas vaginalis detection primers, the front primer sequence is shown in seq ID No.119, and the rear primer sequence is shown in seq ID No.120; Entamoeba histolytica detection primers, the forward primer sequence is shown in seq ID No. 121, and the rear primer sequence is shown in seq ID No. 122; Toxoplasma gondii detection primers, the forward primer sequence is shown in seq ID No.123, and the rear primer sequence is shown in seq ID No.124.

[0009] Optionally, also include: The detection primers of the drug resistance gene 23SrRNA, the forward primer sequence is shown in seq ID No.227, and the rear primer sequence is shown in seq ID No.228; The detection primers of the drug resistance gene parC, the forward primer sequence is shown in seq ID No. 229, and the rear primer sequence is shown in seq ID No. 230; The detection primers of the drug resistance gene 16SrRNA, the forward primer sequence is shown in seq ID No.231, and the rear primer sequence is shown in seq ID No.231; The detection primers of the drug resistance gene penA, the forward primer sequence is shown in seq ID No. 233, and the rear primer sequence is shown in seq ID No. 234; The detection primers of the drug resistance gene ftsX, the forward primer sequence is shown in seq ID No. 235, and the rear primer sequence is shown in seq ID No. 236; The detection primers of the drug resistance gene PBP2x, the forward primer sequence is shown in seq ID No. 237, and the rear primer sequence is shown in seq ID No. 238; The detection primers of the drug resistance gene gyrA, the forward primer sequence is shown in seq ID No. 239, and the rear primer sequence is shown in seq ID No. 240; The detection primers of the drug resistance gene grlA, the forward primer sequence is shown in seq ID No.241, and the rear primer sequence is shown in seq ID No.242; The detection primers of the drug resistance gene graS, the forward primer sequence is shown in seq ID No. 243, and the rear primer sequence is shown in seq ID No. 244; The detection primers of the drug resistance gene rpoB, the forward primer sequence is shown in seq ID No. 245, and the rear primer sequence is shown in seq ID No. 246; The detection primers of the drug resistance gene ntr6, the forward primer sequence is shown in seq ID No. 247, and the rear primer sequence is shown in seq ID No. 248; The detection primers of the drug resistance gene ERG11, the forward primer sequence is shown in seq ID No.249, and the rear primer sequence is shown in seqID No.250.

[0010] Optionally, at least one of the following pathogen detection primers is also included: Prevotella detection primers, the forward primer sequence is shown in seq ID No. 15, and the rear primer sequence is shown in seq ID No. 16; Primers for detecting Enterobacter hallii, the forward primer sequence is shown in seq ID No. 17, and the rear primer sequence is shown in seq ID No. 18; Pseudomonas detection primers, the forward primer sequence is shown in seq ID No. 77, and the rear primer sequence is shown in seq ID No. 78; HPV detection primers: HPV34 detection primers, the forward primer sequence is shown in seq ID No. 195, and the rear primer sequence is shown in seq ID No. 196; HPV44 detection primers, the forward primer sequence is shown in seq ID No. 197, and the rear primer sequence is shown in seq ID No. 198; HPV57 detection primers, the forward primer sequence is shown in seq ID No. 199, and the rear primer sequence is shown in seq ID No. 200; HPV69 detection primers, the forward primer sequence is shown in seq ID No. 201, and the rear primer sequence is shown in seq ID No. 202; HPV71 detection primers, the forward primer sequence is shown in seq ID No. 203, and the rear primer sequence is shown in seq ID No. 204; HPV83 detection primers, the forward primer sequence is shown in seq ID No. 205, and the rear primer sequence is shown in seq ID No. 206; HPV84 detection primers, the front primer sequence is shown in seq ID No.207, and the rear primer sequence is shown in seq ID No.208.

[0011] Optionally, an internal standard sequence is also included, and the nucleic acid sequence of the internal standard sequence has a front primer sequence as shown in seq ID No.251, and a rear primer sequence as shown in seq ID No.252.

[0012] Optionally, the detection primers are all connected to a linker sequence; the nucleic acid sequence of the linker sequence is shown in seq ID No.327 for the front primer sequence and in seq ID No.328 for the rear primer sequence.

[0013] A kit comprises the above-mentioned primer set for high-throughput sequencing of pathogens targeting reproductive tract infections.

[0014] Optionally, the kit further comprises a gDNA digestion reaction reagent, a first-strand synthesis reaction reagent, a first-step amplification reaction reagent, and a second-step amplification reaction reagent; The reagent components of the gDNA digestion reaction include: gDNA wiper Mix reagent; The reagent components of the one-strand synthesis reaction include: RT Mix reagent, Enzyme Mix reagent, nuclease-free water and Random Primers reagent; The reagent components of the first step amplification reaction include: reaction buffer, nuclease-free water, multiplex amplification enzyme and PIC; The reagent components of the second step amplification reaction include: Hifi Mix reagent.

[0015] The primer set for high-throughput sequencing targeting pathogens of reproductive tract infection and the kit are used for high-throughput sequencing to detect pathogens in reproductive tract infection detection samples.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention can detect HPV E6 / E7 mRNA, that is, detect whether the HPV virus is persistently infecting the patient, and provide more accurate clinical triage guidance.

[0017] The detection scope includes common microorganisms in gynecological reproductive tract infections in relatively detail, and accurately classifies them into "pathogenic microorganisms", "suspected human microecological flora", and "beneficial symbiotic bacteria" according to their degree of harm, which helps to achieve accurate diagnosis; the application of this detection technology in the detection of pathogens and drug-resistant genes related to reproductive tract infections has important clinical significance, and can greatly increase the number of detected pathogens and drug-resistant genes in a short period of time, thereby reducing detection costs.

[0018] Based on the above primers, multiplex PCR technology and high-throughput sequencing methods are used to detect pathogens and drug-resistant genes related to reproductive tract infections. Gene fragments of specific detection areas of multiple drug-resistant genes and pathogens are amplified simultaneously. It has strong detection capabilities, high sensitivity, a wide detection range, and a fast speed. The average detection cost of a single gene or pathogen is relatively low. DETAILED DESCRIPTION

[0019] The present invention is further elaborated in detail below in conjunction with specific embodiments. The following embodiments are provided for a better understanding of the present invention, but are not limited to the best implementation mode, and do not limit the content and protection scope of the present invention. Any product that is the same or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the present invention with the features of other prior arts falls within the protection scope of the present invention.

[0020] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the art can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially. The raw materials used are not specifically specified, and they are all commercially available products that can be routinely obtained by those skilled in the art.

[0021] Example 1 Design and obtain primers. The primers required to be designed in this embodiment are primers for detecting E6 / E7 mRNA of HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV67, HPV68, HPV73, and HPV82.

[0022] The method for primer design refers to Chinese patent ZL202411253978.9, and includes the following steps: Step 1) In-depth understanding of the biological characteristics of pathogens or specific gene regions related to the reproductive tract, obtaining the genome data of pathogenic microorganisms, and establishing a pathogenic microorganism genome database.

[0023] Step 2) Compare the homology and differences between different species or strains, and obtain the conserved regions of pathogenic microorganisms based on the pathogenic microorganism genome database.

[0024] Step 3) Design multiple primers based on the conserved regions in step 2), taking into account parameters such as primer length, GC content, and Tm value. Generally speaking, the primer length is usually around 18-25 bases, and the GC content is moderate to ensure stability and specificity. The Tm value should be similar so that amplification can be performed under the same reaction conditions; at the same time, the formation of secondary structures by the primers themselves and the complementarity between primers should be avoided.

[0025] Step 4) Evaluate the specificity and inclusiveness of the multiplex primers designed in step 3) and select multiplex primers with specificity and inclusiveness ≥ 90%. If problems are found, it is necessary to readjust the primer design or further optimize the experimental conditions to ensure that the reproductive tract targeted primers can be accurately and efficiently used for reproductive tract-related detection and research.

[0026] The primers obtained are specifically shown in Table 3. The primer set composed of the above primers can meet the detection requirements of HPV E6 / E7 mRNA under general circumstances.

[0027] Example 2 In this example, based on the primer set of Example 1, primers for detecting E6 / E7 mRNA of HPV6, HPV11, HPV40, HPV42, HPV43, HPV54, HPV61, HPV70, HPV72, HPV81, HPV89, HPV34, HPV44, HPV57, HPV69, HPV71, HPV83, and HPV84 were further designed and added to the primer set of Example 1 to obtain a high-throughput sequencing primer set that can comprehensively detect HPV E6 / E7 mRNA. The obtained primers are specifically shown in Table 3.

[0028] Example 3 In this embodiment, on the basis of the primer set of embodiment 1, the detection primers for pathogens in Table 1 and the drug resistance gene detection primers in Table 2 are further designed and added to the primer set of embodiment 1. The detection range of this primer set includes common microorganisms in gynecological reproductive tract infections in a relatively detailed manner, and accurately classifies them into "pathogenic microorganisms", "suspected human microecological flora", and "beneficial symbiotic bacteria" according to their degree of harm, which helps to achieve accurate diagnosis, greatly increase the number of detected pathogens and drug resistance genes in a short period of time, and reduce detection costs.

[0029] Table 1 List of pathogenic microorganisms

[0030] The designed drug resistance gene detection primers in this embodiment include detection primers for drug resistance gene 23SrRNA, drug resistance gene parC, drug resistance gene 16SrRNA, drug resistance gene penA, drug resistance gene ftsX, drug resistance gene PBP2x, drug resistance gene gyrA, drug resistance gene grlA, drug resistance gene graS, drug resistance gene rpoB, drug resistance gene ntr6, and drug resistance gene ERG11. The various drug resistance genes and their corresponding pathogens and drug resistance gene sites are shown in Table 2.

[0031] Table 2 Drug resistance gene loci

[0032] The detection primers obtained in Examples 1 to 3 are shown in Table 3.

[0033] Table 3 Primer sequences

[0034] Example 4 This embodiment provides a kit for detecting reproductive tract pathogens by high-throughput sequencing using the above primer set. The kit comprises: The primer combination, gDNA digestion reaction, first-strand synthesis reaction, first-step amplification reaction, and reagents for the second-step amplification reaction in Example 3.

[0035] The reagents for the gDNA digestion reaction are: 5× gDNA wiper Mix, nuclease-free water; the reagents for the first-strand synthesis reaction are: 10×RT Mix, 10×Enzyme Mix, Random Primers, nuclease-free water; The reagents for the first step amplification reaction are: 5 × Reaction buffer, multiplex amplification enzyme, PIC; The reagents for the second step amplification reaction are: 2 × Hifi Mix, nuclease-free water, and adapter; The gDNA wiper Mix uses nucleic acid extraction or purification reagents from Guangzhou Heyuan Biotechnology Co., Ltd. (96 servings). RT Mix, Enzyme Mix, and Random Primers are from RNA single-strand synthesis reagent-3 (96 servings) from Guangzhou Dayuanqi Biotechnology Co., Ltd. Reaction buffer, multiplex amplification enzyme, PIC, Hifi Mix, and adapters are from multiplex library buffer-2 (96 servings) from Guangzhou Dayuanqi Biotechnology Co., Ltd. MagPure A3 XP1# (purification magnetic beads), tNGS supporting reagents, reproductive tract pathogen detection panel (MGI platform), and MGI platform library construction index primers (1-24) from Guangzhou Dayuanqi Biotechnology Co., Ltd. are also used in the examples.

[0036] Hifi Mix, adapters, PIC, etc., RNA single-strand synthesis reagent-3 from Guangzhou Dayuanqi Biotechnology Co., Ltd., multiple reconstruction library buffer-2, MagPure A3 XP1# (purification magnetic beads), tNGS supporting reagents, reproductive tract pathogen detection panel (MGI platform), MGI platform library construction index primers (1-24).

[0037] The reaction system of the gDNA digestion reaction is 10 μL, and its composition is as follows: 5× gDNA wiper Mix 2 μL, template not exceeding 400 ng, and nuclease-free water to make up the volume to 10 μL; The reaction system of the one-strand synthesis reaction is 20 μL, and its composition is as follows: 10 μL of gDNA digestion product, 2 μL of 10×RTMix, 2 μL of 10×Enzyme Mix, 1 μL of Random Primers, and 5 μL of nuclease-free water; The amplification reaction system in the first step is divided into a DNA amplification system and an RNA amplification system. The DNA amplification system is 10 μL, and its composition is as follows: 5 × Reaction buffer 2 μL, multiple PCR primers 2 μL, multiple amplification enzyme 0.5 μL, PIC 0.5 μL, nucleic acid extraction product 5 μL; the RNA amplification system is 10 μL, and its composition is as follows: 5 x Reaction buffer 2 μL, multiple PCR primers 2 μL, multiple amplification enzyme 0.5 μL, PIC 0.5 μL, cDNA first-strand product 5 μL; The second step amplification reaction system is 50 μL, and its composition is as follows: 2× Hifi Mix 25 μL, adapter 5 μL, and purified product of the first amplification 20 μL; The procedure of the gDNA digestion reaction was as follows: hot cover at 105°C, 42°C for 2 min, and insulation at 4°C; The procedure of the one-strand synthesis reaction was as follows: hot cover at 105°C, 25°C for 5 min, 42°C for 20 min, 85°C for 5 min, and insulation at 4°C; The procedure of the first step amplification reaction is: 95°C for 10 min, 1 cycle; 95°C for 30 s, 61°C for 2 min, 63°C for 3 min, 65°C for 1 min, 72°C for 1 min, 5 cycles; 95°C for 30 s, 70°C for 3 min, 25 cycles; 4°C insulation; The procedure of the second step amplification reaction is: 98°C 45s, 1 cycle; 98°C 15s, 60°C 30s, 72°C 30s, 6 cycles; 72°C 5min; and 4°C insulation.

[0038] The reagents for gDNA digestion reaction include 5× gDNA wiper Mix; the reagents for one-strand synthesis reaction include 10×RTMix, 10×Enzyme Mix and Random Primers; the reagents for the first step amplification reaction system include 5×Reaction buffer, multiple PCR primers, multiple amplification enzymes and PIC; the reagents for the second step amplification reaction system include 2×Hifi Mix and adapters.

[0039] A method for high-throughput sequencing of targeted reproductive tract infection pathogens based on the above kit comprises the following steps: 1. Co-extraction of pathogen DNA and RNA 1) Take a clean broken tube (MP Lysing Matrix E tube) and add 1000 μL of lysis buffer.

[0040] 2) Mix the sample by oscillation, take 400 µL of the sample and transfer it into the wall breaking tube, and break the wall on the wall breaking instrument: (operating program: vibration intensity (SPEED) M / S=6.0, running time 30 s, pause time 30 s, number of cycles 8).

[0041] 3) Centrifuge at high speed, 12000 rcf, 1 min, take the supernatant (about 1.mL) into a new 2.0mL tube for extraction.

[0042] 4) Add 30 μL of extraction magnetic beads and 40 μL of proteinase K to the 2 ml centrifuge tube containing the sample and vortex to mix.

[0043] 5) Place in a 70℃ constant temperature shaking metal bath for 10 min at 1200 rpm and separate immediately after incubation.

[0044] 6) Place the centrifuge tube on the magnetic rack and carefully remove the liquid with a pipette when the magnetic beads are completely adsorbed.

[0045] 7) Add 800 μL of washing solution, vortex to mix for 1 min, and centrifuge.

[0046] 8) Place the centrifuge tube on the magnetic rack and carefully remove the liquid with a pipette when the magnetic beads are completely adsorbed.

[0047] 9) Add 800 μL of washing solution, vortex to mix for 1 min, and centrifuge.

[0048] 10) Place the centrifuge tube on the magnetic rack. When the magnetic beads are completely adsorbed, carefully remove the liquid with a pipette and repeat this process once.

[0049] 11) Centrifuge for 10 seconds, put the tube back into the tube hole of the magnetic rack, and after all the magnetic beads are adsorbed, use a 10μL gun to remove the liquid at the bottom of the tube, place the centrifuge tube on the magnetic rack, and dry it at room temperature.

[0050] 12) Add 50 μL of elution buffer, vortex to mix, centrifuge, incubate at room temperature for 5 min, and place on a magnetic rack.

[0051] 13) When the magnetic beads are completely adsorbed, transfer the nucleic acid solution to a new 1.5 mL EP tube.

[0052] 2. gDNA Digestion The pathogen DNA and RNA co-extraction products were shaken and mixed, and then centrifuged instantly.

[0053] Measure the DNA concentration of the co-extraction products of pathogen DNA and RNA, and calculate the volumes of "total amount of nucleic acid" and "nuclease-free water" required to be invested in Table 4.

[0054] Prepare the gDNA digestion reaction system, mix gently, and centrifuge briefly. The formula is shown in Table 4: Table 4 gDNA digestion reaction system

[0055] (4) Place the reaction system into a conventional PCR instrument. The reaction program is shown in Table 5: Table 5 gDNA digestion system reaction procedure

[0056] 3. One-strand synthesis Vortex and mix the gDNA digestion product, and centrifuge briefly.

[0057] Prepare a chain synthesis reaction system, mix gently, and centrifuge instantly. The formula is shown in Table 6: Table 6 One-chain synthesis reaction system

[0058] Place the first-strand synthesis reaction system into a conventional PCR instrument, and the reaction program is shown in Table 7: Table 7 Reaction procedures for one-chain synthesis system

[0059] Note: cDNA can be used immediately in multiple amplification experiments; cDNA should avoid repeated freezing and thawing. It is recommended to store it at -20℃ for short-term storage and -70℃ for long-term storage.

[0060] 4. First step PCR amplification This detection method mainly designs multiple PCR specific primers according to the specific regions of pathogens and drug-resistant genes within the detection range, performs PCR amplification, purifies and recovers the amplified products, and then performs high-throughput sequencing. The various primers in the primer set are not specially modified, and the formula and proportion of the reagents used in the PCR reaction can make the PCR amplification conditions of different gene sites consistent, and there is no non-specific amplification in the results. In the first step of PCR amplification, each primer in the amplification primer combination should be connected to a universal sequence containing a linker, and the details of the base sequence are shown in Table 8.

[0061] Table 8 Base sequences of universal adapter sequences for multiplex PCR primers

[0062] The single-strand synthesis product, pathogen DNA and RNA co-extraction product were shaken and mixed, and centrifuged instantly.

[0063] Configure the first step of PCR amplification synthesis reaction system. The amount of primers used in each reaction system is 2µL, and the formulas of the remaining reagents in the system are shown in Table 9 and Table 10: Table 9 DNA amplification system

[0064] Table 10 RNA amplification system

[0065] The first step PCR amplification synthesis reaction system is placed in a conventional PCR instrument, and the reaction program is shown in Table 11: Table 11 First step PCR amplification reaction program

[0066] 5. First step magnetic bead purification 30 minutes before the first round of magnetic bead purification, place the purification magnetic beads at room temperature to allow them to equilibrate to room temperature.

[0067] Combine the first round amplification products of DNA and RNA in one tube, add 30 μL of nuclease-free water, and then add 60 μL of DNA purification magnetic beads; After mixing evenly, let it stand at room temperature for 5 min. After a brief centrifugation, place it on a magnetic rack and let it stand for 3-5 min to separate the magnetic beads.

[0068] When the supernatant is completely clear, remove it.

[0069] Add 200 μL of freshly prepared 80% ethanol to the remaining magnetic beads, let stand at room temperature for 30 seconds, remove the supernatant, and repeat the 80% ethanol wash once.

[0070] After a brief centrifugation, place the beads on a magnetic stand to completely absorb the remaining ethanol and leave them at room temperature for 2-3 minutes to air-dry the beads.

[0071] Add 23 μL of nuclease-free water to resuspend the magnetic beads, place at room temperature for 2 min, centrifuge briefly, and place on a magnetic rack for 2 min to separate the magnetic beads.

[0072] Pipette 20 μL of supernatant into a new 200 μL PCR tube and make sure to mark it. Place the product at 4°C for later use and store it at -20°C for sample storage.

[0073] 6. Second step PCR amplification The first round of magnetic bead purification products of pathogens were shaken and mixed, and then centrifuged briefly.

[0074] Prepare the second step PCR amplification reaction system, mix gently, and centrifuge briefly. The formula is shown in Table 12: Table 12 Second step PCR amplification system

[0075] The second step PCR amplification reaction system was placed in a conventional PCR instrument, and the reaction program was as shown in Table 13: Table 13 First step PCR amplification reaction program

[0076] 7. Second step magnetic bead purification After the second step of PCR amplification, the reaction tube was centrifuged briefly, 50 μL of nuclease-free water was added, and 75 μL of DNA purification magnetic beads were added to the PCR product, mixed, and incubated at room temperature for 5 min; Centrifuge briefly, place the eight-tube strip on a magnetic rack, and transfer the supernatant to a PCR tube containing 15 μL DNA purification magnetic beads after the supernatant is completely clear, mix well, and incubate at room temperature for 5 min.

[0077] Centrifuge briefly, place the eight-tube strip on a magnetic rack, discard the supernatant after it is completely clear, add 200 μL 80% ethanol, let stand for 30 seconds, discard the supernatant, and repeat the 80% ethanol wash once.

[0078] Centrifuge for 5 seconds, place the eight-tube strip on the magnetic rack, and discard the remaining liquid.

[0079] Dry the magnetic beads, add 40 μL of nuclease-free water to resuspend the magnetic beads, mix well, and let stand for 2 minutes.

[0080] After a brief centrifugation, place the eight-tube strip on the magnetic rack and transfer 38 μL of supernatant to a new low-absorption tube.

[0081] The purified product was mixed by vortexing, centrifuged briefly, and then the DNA concentration was measured.

[0082] 8. Second step magnetic bead purification After library amplification, centrifuge the reaction tube briefly, add 50 μL of nuclease-free water, add 75 μL of DNA purification magnetic beads to the PCR product, mix well, and incubate at room temperature for 5 min.

[0083] Centrifuge briefly, place on a magnetic rack, and when the supernatant is completely clear, transfer it to a PCR tube containing 15 μL of DNA purification magnetic beads, mix well, and incubate at room temperature for 5 min.

[0084] Centrifuge briefly, place on a magnetic stand, discard the supernatant when it is completely clear, add 200 μL 80% ethanol, let stand for 30 seconds, discard the supernatant, and repeat the 80% ethanol wash once.

[0085] Centrifuge for 5 seconds, place on a magnetic stand, and discard the remaining liquid.

[0086] Dry the magnetic beads, add 40 μL of nuclease-free water to resuspend the magnetic beads, mix well, and let stand for 2 minutes.

[0087] After a brief centrifugation, place on a magnetic stand and transfer 38 μL of supernatant to a new low-binding tube.

[0088] 9. Measure concentration Measure the DNA concentration of the second step purification product.

[0089] 10. Library mixing and DNB reaction First, mix the outgoing libraries, and then use the MGI DNB kit to perform DNB reaction to obtain the DNB product that can be finally loaded on the machine.

[0090] 11. Sequencing on a high-throughput sequencer According to the instructions, the qualified end products of the DNB reaction were used for sequencing on the MGISEQ series sequencer manufactured by MGI.

[0091] Test Example 1 1. Experimental Methods The high-throughput sequencing method targeting reproductive tract infection pathogens in Example 4 was used to detect common vaginal Gardnerella, beneficial bacteria, suspected human microecological bacteria and drug-resistant genes within the detection range.

[0092] 2. Experimental Results As shown in Table 14, the high-throughput sequencing method targeting reproductive tract infection pathogens in Example 4 can effectively detect Gardnerella vaginalis when testing a sample with a known positive test result for Gardnerella vaginalis.

[0093] Table 14 High-throughput detection results of pathogens of reproductive tract infection

[0094] Test Example 2 1. Experimental Methods Using a high-throughput sequencing method targeting pathogens of reproductive tract infection in Example 4, three clinical specimens of reproductive tract infection were tested, and then the test results were compared with the results of the metagenomic detection method.

[0095] 2. Experimental Results The experimental results are shown in Table 15.

[0096] Table 15 Comparison of high-throughput sequencing and metagenomic detection results of pathogens targeting reproductive tract infection

[0097] Test Example 3 1. Experimental Methods The high-throughput sequencing method targeting pathogens of reproductive tract infection in Example 4 was used to detect three clinical specimens of reproductive tract infection, and the real-time fluorescence PCR method was used to qualitatively detect the E6 / E7 region mRNA of 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) in human cervical exfoliated cells in the above three clinical specimens of reproductive tract infection, and then the detection results of the high-throughput sequencing method were compared with the results of the real-time fluorescence PCR method.

[0098] 2. Experimental Results The experimental results are shown in Table 16.

[0099] Table 16 Comparison of high-throughput sequencing of pathogens of reproductive tract infection and real-time fluorescence PCR detection of mRNA in the E6 / E7 region

[0100] In addition, it should be noted that the above are only preferred embodiments of the present invention and are not limited to the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions recorded in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer set for high-throughput sequencing of pathogens of reproductive tract infection, characterized in that: include: HPV16 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 253, and the rear primer sequence is shown in seq ID No. 254; HPV18 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 255, and the rear primer sequence is shown in seq ID No. 256; HPV26 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 257, and the rear primer sequence is shown in seq ID No. 258; HPV31 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 259, and the rear primer sequence is shown in seq ID No. 260; HPV33 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 261, and the rear primer sequence is shown in seq ID No. 262; HPV35 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 263, and the rear primer sequence is shown in seq ID No. 264; HPV39 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 265, and the rear primer sequence is shown in seq ID No. 266; HPV45 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 267, and the rear primer sequence is shown in seq ID No. 268; HPV51 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 269, and the rear primer sequence is shown in seq ID No. 2700; HPV52 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 271, and the rear primer sequence is shown in seq ID No. 272; HPV53 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 273, and the rear primer sequence is shown in seq ID No. 274; HPV56 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 275, and the rear primer sequence is shown in seq ID No. 276; HPV58 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 277, and the rear primer sequence is shown in seq ID No. 278; HPV59 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 279, and the rear primer sequence is shown in seq ID No. 280; HPV66 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 281, and the rear primer sequence is shown in seq ID No. 282; HPV67 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 283, and the rear primer sequence is shown in seq ID No. 284; HPV68 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 285, and the rear primer sequence is shown in seq ID No. 286; HPV73 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 287, and the rear primer sequence is shown in seq ID No. 288; HPV82 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.289, and the rear primer sequence is shown in seqID No.

290.

2. The primer set for high-throughput sequencing of pathogens targeting reproductive tract infections according to claim 1, characterized in that: Also includes at least one of the following detection primers: HPV6 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 291, and the rear primer sequence is shown in seq ID No. 292; HPV11 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 293, and the rear primer sequence is shown in seq ID No. 294; HPV40 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 295, and the rear primer sequence is shown in seq ID No. 296; HPV42 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 297, and the rear primer sequence is shown in seq ID No. 298; HPV43 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 299, and the rear primer sequence is shown in seq ID No. 300; HPV54 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 301, and the rear primer sequence is shown in seq ID No. 302; HPV61 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 303, and the rear primer sequence is shown in seq ID No. 304; HPV70 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.305, and the rear primer sequence is shown in seq ID No.306; HPV72 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 307, and the rear primer sequence is shown in seq ID No. 308; HPV81 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No. 309, and the rear primer sequence is shown in seq ID No. 310; HPV89 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.311, and the rear primer sequence is shown in seq ID No.312; HPV34 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.313, and the rear primer sequence is shown in seq ID No.314; HPV44 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.315, and the rear primer sequence is shown in seq ID No.316; HPV57 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.317, and the rear primer sequence is shown in seq ID No.318; HPV69 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.319, and the rear primer sequence is shown in seq ID No.320; HPV71 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.321, and the rear primer sequence is shown in seq ID No.322; HPV83 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.323, and the rear primer sequence is shown in seq ID No.324; HPV84 E6 / E7 mRNA detection primers, the forward primer sequence is shown in seq ID No.325, and the rear primer sequence is shown in seqID No.

326.

3. The primer set for high-throughput sequencing of pathogens targeting reproductive tract infections according to claim 1, characterized in that: It also includes at least one of primers for detecting beneficial symbiotic bacteria, primers for detecting anaerobic / facultative anaerobic bacteria, primers for detecting aerobic bacteria, primers for detecting STDs bacteria, primers for detecting mycobacteria, primers for detecting high-risk / suspected high-risk human papillomavirus, primers for detecting low-risk human papillomavirus, primers for detecting other DNA viruses, primers for detecting RNA viruses, primers for detecting fungi, and primers for detecting parasites; and / or The beneficial symbiotic bacteria detection primers include: Primers for detecting Lactobacillus crispatus, the sequence of the front primer is shown in seq ID No.1, and the sequence of the rear primer is shown in seq ID No.2; Primers for detecting Lactobacillus iners, the forward primer sequence is shown in seq ID No.3, and the rear primer sequence is shown in seq ID No.4; The primers for detecting Lactobacillus jensenii, the sequence of the front primer is shown in seq ID No.5, and the sequence of the rear primer is shown in seq ID No.6; Lactobacillus gasseri detection primers, the front primer sequence is shown in seq ID No.7, and the rear primer sequence is shown in seq ID No.8; Lactobacillus detection primers, the forward primer sequence is shown in seq ID No.9, and the rear primer sequence is shown in seq ID No.10; The anaerobic / facultative anaerobic bacteria detection primers include: Primers for detecting Gardnerella vaginalis, the forward primer sequence is shown in seq ID No. 11, and the rear primer sequence is shown in seq ID No. 12; The primers for detecting Atopobium vaginalis, the forward primer sequence is shown in seq ID No. 13, and the rear primer sequence is shown in seq ID No. 14; Primers for detecting Bacteroides fragilis, the forward primer sequence is shown in seq ID No. 19, and the rear primer sequence is shown in seq ID No. 20; Two-way Prevotella detection primers, the forward primer sequence is shown in seq ID No. 21, and the rear primer sequence is shown in seq ID No. 22; Proteus mirabilis detection primers, the forward primer sequence is shown in seq ID No. 23, and the rear primer sequence is shown in seq ID No. 24; Streptococcus agalactiae detection primers, the forward primer sequence is shown in seq ID No. 25, and the rear primer sequence is shown in seq ID No. 26; Streptococcus pneumoniae detection primers, the forward primer sequence is shown in seq ID No. 27, and the rear primer sequence is shown in seq ID No. 28; Anaerobic peptostreptococcus detection primers, the forward primer sequence is shown in seq ID No. 29, and the rear primer sequence is shown in seq ID No. 30; Primers for detecting Fingoldella magna, the forward primer sequence is shown in seq ID No. 31, and the rear primer sequence is shown in seq ID No. 32; Micromonas microti detection primers, the forward primer sequence is shown in seq ID No. 33, and the rear primer sequence is shown in seq ID No. 34; Primers for detecting Clostridium perfringens, the forward primer sequence is shown in seq ID No.35, and the rear primer sequence is shown in seq ID No.36; Mobiluncus timidus detection primers, the forward primer sequence is shown in seq ID No. 37, and the rear primer sequence is shown in seq ID No. 38; Mobiluncus cruzi detection primers, the forward primer sequence is shown in seq ID No.39, and the rear primer sequence is shown in seq ID No.40; The primers for detecting giant cocci, the sequence of the front primer is shown in seq ID No.41, and the sequence of the rear primer is shown in seq ID No.42; Actinomyces ildoni detection primers, the forward primer sequence is shown in seq ID No.43, and the rear primer sequence is shown in seq ID No.44; Oral trichomoniasis detection primers, the forward primer sequence is shown in seq ID No.45, and the rear primer sequence is shown in seq ID No.46; The primers for detecting Megasphaera elsdenii, the sequence of the front primer is shown in seq ID No.47, and the sequence of the rear primer is shown in seq ID No.48; The aerobic bacteria detection primers include: Staphylococcus aureus detection primers, the forward primer sequence is shown in seq ID No. 49, and the rear primer sequence is shown in seq ID No. 50; Primers for detecting Staphylococcus epidermidis, the sequence of the front primer is shown in seq ID No.51, and the sequence of the rear primer is shown in seq ID No.52; Primers for detecting Staphylococcus intermedius, the forward primer sequence is shown in seq ID No.53, and the rear primer sequence is shown in seq ID No.54; Primers for detecting hemolytic Staphylococcus aureus, the forward primer sequence is shown in seq ID No.55, and the rear primer sequence is shown in seq ID No.56; Primers for detecting Staphylococcus lugdunensis, the forward primer sequence is shown in seq ID No.57, and the rear primer sequence is shown in seq ID No.58; Enterococcus faecalis detection primers, the forward primer sequence is shown in seq ID No.59, and the rear primer sequence is shown in seq ID No.60; Enterococcus faecium detection primers, the forward primer sequence is shown in seq ID No.61, and the rear primer sequence is shown in seq ID No.62; Escherichia coli detection primers, the forward primer sequence is shown in seq ID No.63, and the rear primer sequence is shown in seq ID No.64; Klebsiella pneumoniae detection primers, the forward primer sequence is shown in seq ID No.65, and the rear primer sequence is shown in seq ID No.66; Primers for detecting Klebsiella oxytoca, the forward primer sequence is shown in seq ID No.67, and the rear primer sequence is shown in seq ID No.68; The primers for detecting Klebsiella aerogenes, the sequence of the front primer is shown in seq ID No.69, and the sequence of the rear primer is shown in seq ID No.70; Primers for detecting Enterobacter cloacae complex, the forward primer sequence is shown in seq ID No.71, and the rear primer sequence is shown in seq ID No.72; Primers for detecting Citrobacter cohnii, the forward primer sequence is shown in seq ID No.73, and the rear primer sequence is shown in seq ID No.74; Primers for detecting Citrobacter freundii, the forward primer sequence is shown in seq ID No.75, and the rear primer sequence is shown in seq ID No.76; Pseudomonas aeruginosa detection primers, the forward primer sequence is shown in seq ID No.79, and the rear primer sequence is shown in seq ID No.80; Haemophilus influenzae detection primers, the forward primer sequence is shown in seq ID No.81, and the rear primer sequence is shown in seq ID No.82; Haemophilus parainfluenzae detection primers, the forward primer sequence is shown in seq ID No.83, and the rear primer sequence is shown in seq ID No.84; Primers for detecting Streptococcus anginosus, the sequence of the front primer is shown in seq ID No.85, and the sequence of the rear primer is shown in seq ID No.86; The STDs bacteria detection primers include: The primers for detecting Neisseria gonorrhoeae, the sequence of the front primer is shown in seq ID No.87, and the sequence of the rear primer is shown in seq ID No.88; Treponema pallidum detection primers, the front primer sequence is shown in seq ID No.89, and the rear primer sequence is shown in seq ID No.90; Haemophilus ducreyi detection primers, the forward primer sequence is shown in seq ID No.91, and the rear primer sequence is shown in seq ID No.92; Chlamydia trachomatis detection primers, the forward primer sequence is shown in seq ID No.93, and the rear primer sequence is shown in seq ID No.94; Mycoplasma hominis detection primers, the front primer sequence is shown in seq ID No.95, and the rear primer sequence is shown in seq ID No.96; Ureaplasma urealyticum detection primers, the front primer sequence is shown in seq ID No.97, and the rear primer sequence is shown in seq ID No.98; Mycoplasma genitalium detection primers, the front primer sequence is shown in seq ID No.99, and the rear primer sequence is shown in seq ID No.100; The mycobacterium detection primers include: Mycobacterium tuberculosis complex detection primers, the front primer sequence is shown in seq ID No. 103, and the rear primer sequence is shown in seq ID No. 104; The high-risk / suspected high-risk human papillomavirus detection primers: HPV16 detection primers, the forward primer sequence is shown in seq ID No. 135, and the rear primer sequence is shown in seq ID No. 136; HPV18 detection primers, the forward primer sequence is shown in seq ID No. 137, and the rear primer sequence is shown in seq ID No. 138; HPV31 detection primers, the forward primer sequence is shown in seq ID No. 139, and the rear primer sequence is shown in seq ID No. 140; HPV33 detection primers, the forward primer sequence is shown in seq ID No. 141, and the rear primer sequence is shown in seq ID No. 142; HPV35 detection primers, the forward primer sequence is shown in seq ID No. 143, and the rear primer sequence is shown in seq ID No. 144; HPV39 detection primers, the forward primer sequence is shown in seq ID No. 145, and the rear primer sequence is shown in seq ID No. 146; HPV45 detection primers, the forward primer sequence is shown in seq ID No. 147, and the rear primer sequence is shown in seq ID No. 148; HPV51 detection primers, the forward primer sequence is shown in seq ID No. 149, and the rear primer sequence is shown in seq ID No. 150; HPV52 detection primers, the forward primer sequence is shown in seq ID No. 151, and the rear primer sequence is shown in seq ID No. 152; HPV56 detection primers, the forward primer sequence is shown in seq ID No. 153, and the rear primer sequence is shown in seq ID No. 154; HPV58 detection primers, the forward primer sequence is shown in seq ID No. 155, and the rear primer sequence is shown in seq ID No. 156; HPV59 detection primers, the forward primer sequence is shown in seq ID No. 157, and the rear primer sequence is shown in seq ID No. 158; HPV66 detection primers, the forward primer sequence is shown in seq ID No. 159, and the rear primer sequence is shown in seq ID No. 160; HPV68 detection primers, the forward primer sequence is shown in seq ID No. 161, and the rear primer sequence is shown in seq ID No. 162; HPV26 detection primers, the forward primer sequence is shown in seq ID No. 163, and the rear primer sequence is shown in seq ID No. 164; HPV53 detection primers, the forward primer sequence is shown in seq ID No. 165, and the rear primer sequence is shown in seq ID No. 166; HPV67 detection primers, the forward primer sequence is shown in seq ID No. 167, and the rear primer sequence is shown in seq ID No. 168; HPV70 detection primers, the forward primer sequence is shown in seq ID No. 169, and the rear primer sequence is shown in seq ID No. 170; HPV73 detection primers, the forward primer sequence is shown in seq ID No. 171, and the rear primer sequence is shown in seq ID No. 172; HPV82 detection primers, the forward primer sequence is shown in seq ID No. 173, and the rear primer sequence is shown in seq ID No. 174; The low-risk human papillomavirus detection primers include: HPV6 detection primers, the forward primer sequence is shown in seq ID No. 175, and the rear primer sequence is shown in seq ID No. 176; HPV11 detection primers, the forward primer sequence is shown in seq ID No. 177, and the rear primer sequence is shown in seq ID No. 178; HPV40 detection primers, the forward primer sequence is shown in seq ID No. 179, and the rear primer sequence is shown in seq ID No. 180; HPV42 detection primers, the forward primer sequence is shown in seq ID No. 181, and the rear primer sequence is shown in seq ID No. 182; HPV43 detection primers, the forward primer sequence is shown in seq ID No. 183, and the rear primer sequence is shown in seq ID No. 184; HPV54 detection primers, the forward primer sequence is shown in seq ID No. 185, and the rear primer sequence is shown in seq ID No. 186; HPV61 detection primers, the forward primer sequence is shown in seq ID No. 187, and the rear primer sequence is shown in seq ID No. 188; HPV72 detection primers, the forward primer sequence is shown in seq ID No. 189, and the rear primer sequence is shown in seq ID No. 190; HPV81 detection primers, the forward primer sequence is shown in seq ID No. 191, and the rear primer sequence is shown in seq ID No. 192; HPV89 detection primers, the forward primer sequence is shown in seq ID No. 193, and the rear primer sequence is shown in seq ID No. 194; Other DNA virus detection primers include: Human herpes virus type I detection primers, the front primer sequence is shown in seq ID No. 209, and the rear primer sequence is shown in seq ID No. 210; Human herpes virus type II detection primers, the front primer sequence is shown in seq ID No.211, and the rear primer sequence is shown in seq ID No.212; Epstein-Barr virus detection primers, the forward primer sequence is shown in seq ID No. 213, and the rear primer sequence is shown in seq ID No. 214; Cytomegalovirus detection primers, the forward primer sequence is shown in seq ID No. 215, and the rear primer sequence is shown in seq ID No. 216; Primers for detecting molluscum contagiosum virus, the sequence of the front primer is shown in seq ID No. 217, and the sequence of the rear primer is shown in seq ID No. 218; Hepatitis B virus detection primers, the front primer sequence is shown in seq ID No. 219, and the rear primer sequence is shown in seq ID No. 220; Human polyomavirus type 1 detection primers, the forward primer sequence is shown in seq ID No. 221, and the rear primer sequence is shown in seq ID No. 222; Human polyomavirus type 2 detection primers, the forward primer sequence is shown in seq ID No. 223, and the rear primer sequence is shown in seq ID No. 224; Varicella-zoster virus detection primers, the forward primer sequence is shown in seq ID No. 225, and the rear primer sequence is shown in seq ID No. 226; RNA virus detection primers include: Human immunodeficiency virus type I detection primers, the forward primer sequence is shown in seq ID No.125, and the rear primer sequence is shown in seq ID No.126; Human immunodeficiency virus type II detection primers, the forward primer sequence is shown in seq ID No.127, and the rear primer sequence is shown in seq ID No.128; Hepatitis A virus detection primers, the front primer sequence is shown in seq ID No.129, and the rear primer sequence is shown in seq ID No.130; Hepatitis C virus detection primers, the front primer sequence is shown in seq ID No.131, and the rear primer sequence is shown in seq ID No.132; Human T-lymphotropic virus type 1 detection primers, the forward primer sequence is shown in seq ID No.133, and the rear primer sequence is shown in seq ID No.134; The fungus detection primers include: Candida albicans detection primers, the forward primer sequence is shown in seq ID No. 105, and the rear primer sequence is shown in seq ID No. 106; Primers for detecting Candida tropicalis, the sequence of the front primer is shown in seq ID No. 107, and the sequence of the rear primer is shown in seq ID No. 108; Primers for detecting Candida glabrata, the sequence of the front primer is shown in seq ID No. 109, and the sequence of the rear primer is shown in seq ID No. 110; Candida krusei detection primers, the forward primer sequence is shown in seq ID No.111, and the rear primer sequence is shown in seq ID No.112; Primers for detecting Portuguese Candida albicans, the sequence of the front primer is shown in seq ID No. 113, and the sequence of the rear primer is shown in seq ID No. 114; Primers for detecting Candida parapsilosis, the sequence of the front primer is shown in seq ID No.115, and the sequence of the rear primer is shown in seq ID No.116; Primers for detecting Candida dulio, the forward primer sequence is shown in seq ID No.117, and the rear primer sequence is shown in seq ID No.118; The parasite detection primers include: Trichomonas vaginalis detection primers, the front primer sequence is shown in seq ID No.119, and the rear primer sequence is shown in seq ID No.120; Entamoeba histolytica detection primers, the forward primer sequence is shown in seq ID No. 121, and the rear primer sequence is shown in seq ID No. 122; Toxoplasma gondii detection primers, the forward primer sequence is shown in seq ID No.123, and the rear primer sequence is shown in seq ID No.

124.

4. The primer set for high-throughput sequencing of pathogens targeting reproductive tract infections according to claim 1, characterized in that: Also includes: The detection primers of the drug resistance gene 23SrRNA, the forward primer sequence is shown in seq ID No.227, and the rear primer sequence is shown in seqID No.228; The detection primers of the drug resistance gene parC, the forward primer sequence is shown in seq ID No. 229, and the rear primer sequence is shown in seq ID No. 230; The detection primers of the drug resistance gene 16SrRNA, the forward primer sequence is shown in seq ID No.231, and the rear primer sequence is shown in seqID No.231; The detection primers of the drug resistance gene penA, the forward primer sequence is shown in seq ID No. 233, and the rear primer sequence is shown in seq ID No. 234; The detection primers of the drug resistance gene ftsX, the forward primer sequence is shown in seq ID No. 235, and the rear primer sequence is shown in seq ID No. 236; The detection primers of the drug resistance gene PBP2x, the forward primer sequence is shown in seq ID No. 237, and the rear primer sequence is shown in seq ID No. 238; The detection primers of the drug resistance gene gyrA, the forward primer sequence is shown in seq ID No. 239, and the rear primer sequence is shown in seq ID No. 240; The detection primers of the drug resistance gene grlA, the forward primer sequence is shown in seq ID No. 241, and the rear primer sequence is shown in seq ID No. 242; The detection primers of the drug resistance gene graS, the forward primer sequence is shown in seq ID No. 243, and the rear primer sequence is shown in seq ID No. 244; The detection primers of the drug resistance gene rpoB, the forward primer sequence is shown in seq ID No. 245, and the rear primer sequence is shown in seq ID No. 246; The detection primers of the drug resistance gene ntr6, the forward primer sequence is shown in seq ID No. 247, and the rear primer sequence is shown in seq ID No. 248; The detection primers of the drug resistance gene ERG11, the forward primer sequence is shown in seq ID No.249, and the rear primer sequence is shown in seq ID No.

250.

5. The primer set for high-throughput sequencing of pathogens targeting reproductive tract infections according to claim 1, characterized in that: Also includes at least one of the following pathogen detection primers: Prevotella detection primers, the forward primer sequence is shown in seq ID No. 15, and the rear primer sequence is shown in seq ID No. 16; Primers for detecting Enterobacter hallii, the forward primer sequence is shown in seq ID No. 17, and the rear primer sequence is shown in seq ID No. 18; Pseudomonas detection primers, the forward primer sequence is shown in seq ID No.77, and the rear primer sequence is shown in seq ID No.78; HPV detection primers: HPV34 detection primers, the forward primer sequence is shown in seq ID No. 195, and the rear primer sequence is shown in seq ID No. 196; HPV44 detection primers, the forward primer sequence is shown in seq ID No. 197, and the rear primer sequence is shown in seq ID No. 198; HPV57 detection primers, the forward primer sequence is shown in seq ID No. 199, and the rear primer sequence is shown in seq ID No. 200; HPV69 detection primers, the forward primer sequence is shown in seq ID No. 201, and the rear primer sequence is shown in seq ID No. 202; HPV71 detection primers, the forward primer sequence is shown in seq ID No. 203, and the rear primer sequence is shown in seq ID No. 204; HPV83 detection primers, the forward primer sequence is shown in seq ID No. 205, and the rear primer sequence is shown in seq ID No. 206; HPV84 detection primers, the front primer sequence is shown in seq ID No.207, and the rear primer sequence is shown in seq ID No.

208.

6. The primer set for high-throughput sequencing of pathogens targeting reproductive tract infections according to claim 1, characterized in that: It also includes an internal standard sequence, the nucleic acid sequence of the internal standard sequence has a front primer sequence as shown in seq ID No.251, and a rear primer sequence as shown in seq ID No.

252.

7. The primer set for high-throughput sequencing of pathogens targeting reproductive tract infections according to claim 1, characterized in that: The detection primers are all connected with a linker sequence; the nucleic acid sequence of the linker sequence is shown in seq ID No.327 for the front primer sequence and in seq ID No.328 for the rear primer sequence.

8. A kit, characterized in that: A primer set for high-throughput sequencing of pathogens targeting reproductive tract infections comprising any one of claims 1 to 7.

9. The kit according to claim 8, characterized in that It also includes gDNA digestion reaction reagents, one-strand synthesis reaction reagents, first-step amplification reaction reagents, and second-step amplification reaction reagents; The reagent components of the gDNA digestion reaction include: gDNA wiper Mix reagent; The reagent components of the one-strand synthesis reaction include: RT Mix reagent, Enzyme Mix reagent, nuclease-free water and Random Primers reagent; The reagent components of the first step amplification reaction include: reaction buffer, nuclease-free water, multiplex amplification enzyme and PIC reagent; The reagent components of the second step amplification reaction include: Hifi Mix reagent.

10. Use of the primer set for high-throughput sequencing targeting pathogens of reproductive tract infection according to any one of claims 1 to 7 and the kit according to claim 8 or 9 for high-throughput sequencing detection of pathogens in reproductive tract infection test samples.

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