Primer combination for identifying six genital tract pathogens and detecting drug-resistant genes, kit and application

Abstract

The invention discloses a primer combination for identifying six genital tract pathogens and detecting drug resistance genes, a kit and application, the primer combination comprises a primer combination for identifying chlamydia trachomatis, mycoplasma genitalium, ureaplasma urealyticum, mycoplasma hominis, neisseria gonorrhoeae and trichomonas vaginalis and detecting the drug resistance genes of the six genital tract pathogens, and nucleotide sequences are shown as SEQ ID NO.1-SEQ ID NO.50. Microorganisms and related drug-resistant genes in a detection range are identified by utilizing a multiple PCR targeted three-generation sequencing method, the specificity is good, the sensitivity is high, the detection technology has important clinical significance when being applied to identification of genital tract infection pathogens, and compared with a traditional detection method, the detection flux, the coverage range and the sensitivity are higher, and detection is stable; compared with pathogenic microorganism metagenome (mNGS) detection, the method has the advantages that the detection cost is lower, and the detection sensitivity on drug-resistant genes and drug-resistant gene mutation is higher.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a primer combination, a kit and applications for the identification of six reproductive tract pathogens and the detection of drug-resistant genes. Background Art

[0002] Most infections with curable sexually transmitted infections are asymptomatic. Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Trichomonas vaginalis, and Mycoplasma genitalium are the leading causes of sexually transmitted infections (STIs) worldwide. Resistance of Mycoplasma genitalium to first-line macrolide antibiotics and second-line quinolones is increasing globally. Therefore, accurate diagnosis is crucial for interrupting transmission chains and implementing targeted treatment.

[0003] Current diagnostic methods for sexually transmitted infection pathogens include microbial culture, direct microscopy, serological testing, direct immunofluorescence assays, and nucleic acid amplification tests (NAATs). Bacterial culture is suitable for diagnosing pathogens such as Neisseria gonorrhoeae and Chlamydia trachomatis, but it is difficult to culture most pathogens in vitro and typically requires more than 2-3 days. Direct microscopy primarily identifies common pathogens based on their typical biological morphology. While rapid, it relies on subjective testing, resulting in poor reproducibility, low accuracy, and insufficient sensitivity (for example, the detection rate for Trichomonas vaginalis is 50%-60%), which can lead to missed detections. Serological immunoassays are convenient and require less specialized equipment and personnel, but they are also less accurate and prone to false negatives. Furthermore, these traditional testing techniques cannot meet clinical needs for detecting complex infections and for simultaneous detection of drug-resistance genes.

[0004] In contrast, nucleic acid amplification testing has become the new "gold standard" for diagnosing a variety of sexually transmitted infections due to its rapidity, high sensitivity, and specificity. This technology also enables the simultaneous detection of multiple pathogens. Nucleic acid testing has a good balance between cost, detection speed, and detection performance, making it a mainstream detection method. Despite advances in nucleic acid amplification detection technology, the drug resistance of drug-resistant pathogens is often related to gene mutations, which can be detected through molecular biology techniques. However, existing nucleic acid amplification tests cannot fully complete the diagnosis of drug resistance, and related mutation analysis still relies on gene sequencing. In order to develop precise treatment plans, nucleic acid amplification testing needs to be combined with gene sequencing, resulting in a time-consuming diagnostic process. Therefore, there is an urgent need to develop new molecular detection technologies that can simultaneously and rapidly detect pathogens and their resistance-related mutations.

[0005] To address these challenges, targeted next-generation sequencing (tNGS) technology has demonstrated breakthrough advantages. This technology uses ultra-multiplex PCR to enrich hundreds of pathogens and drug resistance / virulence genes in clinical samples, combining high-throughput sequencing with database comparison to rapidly complete pathogen spectrum analysis. Compared to traditional methods, tNGS offers the following significant advantages: ① High-throughput coverage: A single test can identify multiple pathogens of reproductive tract infections; ② Accurate early warning of drug resistance: Simultaneous analysis of key mutations, such as the metronidazole resistance gene in Trichomonas vaginalis and the tetracycline resistance gene in Chlamydia trachomatis; ③ Time advantage: The simultaneous detection of resistance genes and identification genes can significantly shorten the time required for clinical diagnosis and treatment reporting; ④ Clinical decision support: tNGS provides pathogen composition analysis, drug resistance prediction, and treatment plan recommendations.

[0006] Nanopore sequencing is a single-molecule, real-time sequencing technology that also does not rely on traditional microbial culture and can rapidly detect suspected pathogens in clinical samples. It uses the changes in current as single molecules of DNA or RNA in a sample pass through a nanopore to record the base composition and sequence the entire genome or sequence. The main advantage of nanopore sequencing is its ultra-long read length, which can produce sequences ranging from 1kb to over 100kb. Longer sequences offer greater advantages in analyzing pathogens and resistance / virulence genes. Another advantage is that it can begin acquiring sequencing data within seconds of adding the sample, enabling real-time genome alignment, thereby quickly obtaining species information of the microorganisms in the sample and information on the identification / resistance genes they carry. In other words, when nanopore sequencing is combined with efficient genomic nucleic acid extraction and library construction methods, test results can be obtained within 6-7 hours, thereby enabling rapid diagnosis and treatment of patients in clinics.

[0007] The emerging multiplex targeted amplification sequencing technology can solve the detection scope and coverage. However, some patents provide relatively few primer combinations and detect a single type of reproductive tract pathogens, such as application number CN202010809876.6, a method for multiple rapid sequencing of gonococcal resistance genes, which discloses the use of nanopore technology to identify and detect Neisseria gonorrhoeae; although some patents have a relatively comprehensive detection range, such as application number CN202510283181.1, a primer set, kit and application for high-throughput sequencing of pathogens of reproductive tract infections, which discloses the use of multiplex PCR combined with high-throughput sequencing to identify microorganisms and related resistance genes within the detection range, the sequencing technology is second-generation sequencing, and there are difficulties in species identification for mixed infections, such as the 23S resistance gene with extremely high homology, especially the inability to determine the resistance of the corresponding pathogens in mixed infections.

[0008] Therefore, looking at the existing tNGS technology sequencing product market, there are still key shortcomings: current development focuses mainly on respiratory pathogens, and dedicated detection systems for reproductive tract infection pathogens and drug-resistant genes are not yet mature, especially for the technical difficulties such as the precise positioning of drug-resistant mutations in various reproductive tract mycoplasmas and chlamydia. This technological gap restricts the application potential of this technology in the prevention and control of sexually transmitted infections. Therefore, the present invention combines third-generation nanopore sequencing technology to develop a detection kit for common reproductive tract STI infection pathogens and drug-resistant genes to fill the technical gap in the market. Summary of the Invention

[0009] In order to solve at least one of the above problems, the present invention provides a primer combination, a kit and an application for the identification of six reproductive tract pathogens and the detection of drug-resistant genes.

[0010] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides six primer combinations for identifying reproductive tract pathogens and detecting drug-resistant genes, the primer combinations comprising: (1) The primer combination for detecting Chlamydia trachomatis and its drug-resistant genes includes four pairs of primers: Primer01_R / F-Primer04_R / F, and the nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.8; (2) The primer combination for the detection of Mycoplasma genitalium and its drug-resistant genes includes four pairs of primers: Primer05_R / F-Primer08_R / F, and the nucleotide sequences are shown in SEQ ID NO.9-SEQ ID NO.16; wherein, in the Primer08_F sequence, the base "C" at position 21 and the base "G" at position 22 are both locked nucleic acids, and in the Primer08_R sequence, the base "C" at position 21 and the base "G" at position 23 are both locked nucleic acids; (3) The primer combination for the detection of Ureaplasma urealyticum and its drug-resistant genes includes five pairs of primers: Primer09_R / F-Primer13_R / F, and the nucleotide sequences are shown in SEQ ID NO.17-SEQ ID NO.26; among them, the base "G" at position 20 in the Primer13_F sequence is a locked nucleic acid; (4) The primer combination for the detection of Mycoplasma hominis and its drug-resistant genes includes four pairs of primers: Primer14_R / F-Primer17_R / F, and the nucleotide sequences are shown in SEQ ID NO.27-SEQ ID NO.34; wherein, in the Primer14_R sequence, the 18th base "C" is a locked nucleic acid; in the Primer16_R sequence, the 19th base "C" is a locked nucleic acid; (5) The primer combination for detecting Neisseria gonorrhoeae and its drug-resistant genes includes five pairs of primers: Primer18_R / F-Primer22_R / F, and the nucleotide sequences are shown in SEQ ID NO.35-SEQ ID NO.44; (6) The primer combination for the detection of Trichomonas vaginalis and its drug-resistant genes includes three pairs of primers: Primer23_R / F and Primer25_R / F, and the nucleotide sequences are shown in SEQ ID NO.45-SEQ ID NO.50.

[0011] In some embodiments of the present invention, the drug resistance gene comprises: (1) Targeting the A2057G, A2058C, A2059G, and T2611C ​​sites of the macrolide resistance gene 23S of Chlamydia trachomatis; targeting the S83I and S83R sites of the quinolone resistance gene gyrA; and targeting the R83G site of the quinolone resistance gene ParC; (2) Targeting the A2058G, A2058C, A2058T, and A2059G sites of the macrolide resistance gene 23S of Mycoplasma genitalium; targeting the M95I and D99N sites of the quinolone resistance gene gyrA; and targeting the S83R, S83I, S83N, D87N, D87Y, D87H, and D87G sites of the quinolone resistance gene ParC; (3) Targeting the G2654T and T2245C sites of the macrolide resistance gene 23S of Ureaplasma urealyticum; targeting the Q104K and D112E sites of the quinolone resistance gene gyrA; targeting the S83L, E87Q, E87K, and D82N sites of the quinolone resistance gene ParC; targeting the C1192T, G966T, and G967T sites of the tetracycline resistance gene 16S for plasmid-mediated resistance of the tetracycline resistance gene tetM; (4) Targeting the A2058G, A2059G, and G2057A sites of the macrolide resistance gene 23S of Mycoplasma hominis; targeting the S153A and S153L sites of the quinolone resistance gene gyrA; and targeting the S91I site of the quinolone resistance gene ParC; (5) Targeting the A2059G and T2611G sites of the macrolide resistance gene 23S of Neisseria gonorrhoeae; targeting the S91F, D95Y, D95A, D95G, and D95N sites of the quinolone resistance gene gyrA; targeting the D86N, S88A, S88P, E91A, E91G, E91K, and E91Q sites of the quinolone resistance gene ParC; and targeting the D345 aminoacid insertion, A501T, and G542S sites of the penicillin resistance gene PenA; (6) Targeting the Y71Stop site of the metronidazole resistance gene Ntr4 and the K80Stop site of Ntr6 of Trichomonas vaginalis.

[0012] In some embodiments of the present invention, the concentration ratio of each primer in the primer combination is: Primer01_R / F:Primer02_R / F:Primer03_R / F:Primer04_R / F:Primer05_R / F:Primer06_R / F:Primer07_R / F:Pr imer08_R / F:Primer09_R / F:Primer10_R / F:Primer11_R / F:Primer12_R / F:Primer13_R / F:Primer14_R / F:Prime r15_R / F: Primer16_R / F: Primer17_R / F: Primer18_R / F: Primer19_R / F: Primer20_R / F: Primer21_R / F: Primer22 _R / F: Primer23_R / F: Primer24_R / F: Primer25_R / F=3:2:2:4:4:4:4:3:3:3:4:4:4:4:2:2:3:3:4:2:2:3:4:4:3.

[0013] The second aspect of the present invention provides the use of the primer combination described in the first aspect in the preparation of a product for detecting the identification and drug resistance genes of six reproductive tract pathogens.

[0014] The third aspect of the present invention provides a kit for identifying six reproductive tract pathogens and detecting drug-resistant genes, wherein the kit comprises the primer combination described in the first aspect.

[0015] In some embodiments of the present invention, the kit further includes sample nucleic acid extraction reagents, PCR amplification reagents, and nanopore sequencing reagents.

[0016] A fourth aspect of the present invention provides a method for identifying six reproductive tract pathogens and detecting drug-resistant genes for non-diagnostic and non-therapeutic purposes, the method using the kit described in the third aspect, comprising the following steps: S1, pre-treatment of the sample to be tested; S2. Extracting nucleic acid from the sample to be tested using a nucleic acid extraction reagent; S3, preparing a PCR reaction system using the extracted nucleic acid of the sample to be tested and a PCR amplification reagent, and performing multiplex PCR amplification; S4. After the amplified product is purified, the library is constructed using nanopore sequencing reagents; S5. Perform high-throughput sequencing on the constructed library.

[0017] In some embodiments of the present invention, the final concentration of the primer set in the PCR reaction system is 0.1 μM to 0.5 μM; the final concentration of the sample DNA is greater than or equal to 0.2 ng / μL; and the final concentration of the multiplex reaction amplification enzyme is 0.05 U / μL.

[0018] In some specific embodiments of the present invention, the PCR reaction system contains 4 volumes of nucleic acid, 2.8 volumes of primer mixture, 20 volumes of 2× PCR buffer, 0.2 volumes of multiplex amplification enzyme, 13 volumes of double-distilled water, and a total volume of 40 volumes.

[0019] In some embodiments of the present invention, the amplification conditions of the PCR amplification are 94° C. for 2 min; 94° C. for 30 s, 60° C. for 90 s, 72° C. for 90 s, 34 cycles; 72° C. for 10 min, and 12° C. hold.

[0020] In some embodiments of the present invention, a data analysis step is also included: after logging off the machine, the qualified sequence is compared with the set reference sequence to obtain the mutation status of the common drug resistance mutation sites of the reproductive tract pathogen identification gene and the drug resistance gene in the sample.

[0021] In some embodiments of the present invention, the pre-treatment method of the sample to be tested is: (1) For liquid samples, centrifuge the sample at 12,000 rpm for 5 minutes, discard an appropriate amount of supernatant, and keep about 400 μL of liquid, then pipette and mix thoroughly; (2) If it is a tissue sample, grind the tissue sample with an electric grinder, make up to 1 mL with water and transfer it to a new grinding tube. Centrifuge at 12,000 rpm for 5 minutes, discard an appropriate volume of supernatant, leaving about 400 μL of liquid, and mix it by pipetting.

[0022] In some embodiments of the present invention, the tissue sample is an attachment carrier, which is a daily necessity that absorbs or adheres to genital secretions, including: (1) personal hygiene products: sanitary napkins, sanitary napkins, and panty liners; (2) underwear: underwear and briefs; (3) disposable items: sanitary paper towels and sampling swabs; (4) other carriers that may retain secretions: bed sheets, clothing fibers, etc.

[0023] In some embodiments of the present invention, the high-throughput sequencing is third-generation nanopore sequencing, and there are multiple choices for sequencing chips and sequencing instruments, including sequencing chips and sequencing instruments from Oxford Nanopore in the UK or domestic instruments such as BGI; sequencing kits: SQK-LSK109; SQK-LSK110, sequencing chips: R9 / R10 for nanosequencer sequencing; there are multiple choices for open source software, including centrifuge, minimap2, and TBProfiler.

[0024] In some embodiments of the present invention, the detection method is applicable to the purpose of disease diagnosis and treatment, and is also applicable to non-disease diagnosis and treatment purposes, including clinical diagnosis scenarios and laboratory detection scenarios, including: in hospitals, clinics and other medical institutions, testing of secretions directly collected from patients or their attached carriers; in scientific research or disease control agencies, batch testing of epidemiological survey samples (such as secretion carriers for population screening) based on public health needs.

[0025] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention successfully established a method for detecting six STI infection pathogens: Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and their resistance gene mutation sites using a multiplex PCR targeted third-generation sequencing method. In the reaction system prepared by the present invention, the designed specific primers were modified and optimized with locked nucleic acid to increase the compatibility with the Tm value of other primer systems in the multiplex reaction. After screening, the primers can specifically and sensitively bind to the corresponding pathogen nucleic acid sequence. After the primer concentration is optimized, the amplification efficiency is close. Compared with traditional detection methods, the detection throughput, coverage and sensitivity are higher and the detection is stable; compared with pathogenic microorganism metagenomic (mNGS) detection, the detection cost is lower and the detection sensitivity of resistance genes and resistance gene mutations is higher.

[0026] (2) The present invention can simultaneously detect up to 20 different types of drug-resistant genes; and the sequencing data length is between 600 and 1000 bp. Compared with the second-generation platform, it can greatly improve the accuracy and stability of pathogen identification, and provide a more effective and rapid means for the detection of pathogens of reproductive tract STI infection and their drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 An example of screening specific primers for amplifying Chlamydia trachomatis drug resistance genes in Example 2 of the present invention is shown; Figure 2 An example of screening primers for the drug-resistant gene 23S of Mycoplasma genitalium in Example 2 of the present invention is shown. DETAILED DESCRIPTION

[0028] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0030] The technical solution of the present application will be further described in detail below in conjunction with specific implementation methods.

[0031] Example 1 Detection method process establishment 1. Sample pretreatment If the sample is liquid, centrifuge it at 12,000 rpm for 5 minutes, discard an appropriate amount of supernatant, leaving about 400 μL of residual liquid, and mix thoroughly by pipetting. If the sample is a tissue, grind it with an electric grinder, make up to 1 mL with water and transfer it to a new grinding tube. Centrifuge at 12,000 rpm for 5 minutes, discard an appropriate amount of supernatant, leaving about 400 μL of residual liquid, and mix thoroughly by pipetting.

[0032] 2. Nucleic Acid Extraction Commercial kits were used for nucleic acid extraction, and the extraction method was carried out according to the kit instructions.

[0033] 3. Multiplex Targeted Amplification The extracted sample DNA was subjected to multiplex PCR amplification. The PCR amplification system included a multiplex amplification primer mixture composed of 25 primer pairs, with each primer concentration ranging from 0.1 nM to 0.5 nM; a multiplex reaction amplification enzyme concentration of 0.1 U / 20 μL; the sample DNA to be tested served as a template; the multiplex PCR reaction system with a total volume of 40 μL was shown in Table 1, and the reaction procedure was shown in Table 2.

[0034] Table 1 Multiplex PCR amplification reaction conditions

[0035] Table 2 Multiplex PCR amplification reaction program

[0036] In the multiplex targeted amplification step, in order to efficiently amplify gene fragments, takaraRR062A can be selected as the multiplex reaction amplification enzyme.

[0037] 4. Library Construction (1) End repair: The amplification products of the previous multiple targeted amplification were purified by magnetic beads, and the elution volume of the purified DNA solution was 50 μL; the end repair reaction solution was prepared in a 0.2 mL PCR tube. The end repair reaction system was shown in Table 3, and the reaction conditions were shown in Table 4. Gently stir to mix.

[0038] Table 3 End repair reaction system

[0039] Table 4 End repair reaction procedure

[0040] (2) Barcode ligation: Introduce the barcode tag sequence through ligation reaction, that is, each sample corresponds to a unique barcode tag sequence; prepare the barcode ligation reaction solution in a 0.2 mL PCR tube. The barcode ligation reaction system is shown in Table 5, and the reaction conditions are shown in Table 6. Gently stir to mix; Table 5 Barcode ligation reaction system

[0041] Table 6 Barcode ligation reaction procedure

[0042] (3) Adapter ligation reaction: All sample products were mixed into one tube and then purified again by magnetic beads, and eluted with 35 μL of water. After purification, the motor protein and sequencing adapter were added by ligation reaction to form a sequencing library. The adapter ligation reaction system is shown in Table 7. Gently stir to mix. Table 7 Barcode ligation reaction system

[0043] Ligation reaction procedure: 20°C for 20 min; (4) Prepare the library for the machine: Purify the product again with magnetic beads and elute with 15 μL EB; prepare the library for the machine, the reaction system for the library for the machine is shown in Table 8, and gently stir to mix; Table 8 Reaction system for on-machine library

[0044] (5) Chip preparation: Take 1 mL of FB, add 30 μL of FLT, shake and mix, open the chip Priming port, remove bubbles before using the chip, then inject 800 μL (FB + FLT), let it stand at room temperature for 5 minutes, open the Spoton sample port, and add another 200 μL (FB + FLT) mixture from the Priming port; (6) Sample loading: Mix and suspend the library described in step 5) by pipetting, and slowly drip it into the chip from the Spoton sample port. Cover the Priming port and Spoton sample port, and aspirate the waste liquid from Waste port 1; (7) Sequencing: Set up the sequencing software parameters according to the laboratory requirements and click Start Run to start sequencing.

[0045] This kit is compatible with a variety of sequencing products and reagents. Sequencing chips and instruments are available, including those from Oxford Nanopore (UK) or domestic instruments from BGI. Sequencing kits include SQK-LSK109 and SQK-LSK110, and sequencing chips include R9 / R10 for Nanosequencer sequencing. Open-source software options include Centrifuge, Minimap2, and TBProfiler.

[0046] (8) Data Analysis: After the machine is off, the qualified sequence is compared with the set reference sequence to obtain the mutation status of the common drug-resistant mutation sites of the genital tract STI infection pathogen identification genes and drug-resistant genes in the sample. The information of the common drug-resistant mutation sites of the genital tract STI infection pathogen identification genes and drug-resistant genes is shown in Table 9.

[0047] Table 9 Common drug-resistant mutation sites of genes identifying pathogens of reproductive tract STI infection and drug-resistant genes

[0048] Example 2 Primer design and screening As a multiplex amplification system, the identification / drug resistance amplification system of this system requires consideration of not only the amplification efficiency of individual primers but also the compatibility of each primer within the system, as well as matching the sequencing platform. This example screened the optimal targeted amplification region for the genes identified in Example 1 based on the gene sequence. Targeted primers were designed, screened, and filtered based on the characteristics of the nanopore sequencing platform, resulting in comprehensive primer sequence optimization.

[0049] Primer design includes optimization of both the bioinformatics end and the wet experiment end.

[0050] 1. Collect DNA sequences of target genes of reproductive tract STI infection pathogens and drug resistance genomes and construct a database This database contains reference genome sequences for the construction of specific primers for six STI pathogens: Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis. These sequences were primarily obtained from multiple authoritative databases, including the NCBINT database (ftp: / / ftp.ncbi.nlm.nih.gov / blast / db / FASTA / nt.gz), GenBank (ftp: / / ftp.ncbi.nlm.nih.gov / genomes), and the Parasite Reference Sequence Database (https: / / eupathdb.org / eupathdb). The database contains comprehensive and complete genome sequences for all six STI pathogens, removing redundant, repetitive, and low-reliability sequences to ensure the integrity, accuracy, and comprehensiveness of the microbial sequences. This reduces sequence redundancy and improves the quality of primer sequence design and specificity assessment.

[0051] 2. Specific primer design Based on the above-constructed genomic database of six STI infection pathogens, including Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis, the primer3+blast method was used to predict and screen the initial primer sequences with the highest inclusiveness and best species specificity from the genomic database to form the initial primer pool. The amplicon read length was limited to 600-1200bp, and the Tm value was limited to 59-61°C.

[0052] 3. Primer selection and pooling Primer pairs were comprehensively evaluated and selected based on primer recognition, coverage, melting temperature deviation, number of complementary bases in nonspecific amplification, and dimer ΔG value.

[0053] ① Use MFEprimer v3.2.6 to predict the probability of dimer formation for all primer pools, and only retain dimers with a dimer score greater than or equal to 10; ② Calculate the network relationship of all primer-dimer pairings and sort them from high to low according to the number of dimers formed; ③ Remove the primer with the highest number of dimers from the primer pool, and recalculate the network relationship until each primer pair forms no more than three pairs of dimers, completing dynamic screening; ④ Finally, manually screen each species identification and drug resistance gene primer based on the scoring indicators of various primer design parameters.

[0054] 4. Wet test verification and optimization (1) Through the above-mentioned bioinformatics optimization, multiple sets of primer sequences are preliminarily designed, which are then used for wet experiments for verification. Based on the amplification results of the wet experiments, the primers are designed again through bioinformatics analysis, and finally the optimal set of single primers is obtained.

[0055] Taking the amplification primers of Chlamydia trachomatis resistance genes as an example, the Chlamydia trachomatis resistance genes are gyrA gene, parC gene, and 23S gene, with theoretical lengths of 672bp, 773bp, and 964bp, respectively; through wet experiment amplification verification, it can be seen that the specific Figure 1 As shown in the figure, M is Marker, and the Marker range is 200bp-2000bp; from left to right, they represent Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), Neisseria gonorrhoeae (NG), other common genital tract bacteria and fungi (MIX), and negative control (NC). The product bands after nucleic acid amplification using Chlamydia trachomatis resistance gene amplification primers can be seen from the results. The primer amplification length is consistent with the theoretical amplification length, and the band is single and bright, and there is no specific cross-reaction to other common genital tract pathogens and microorganisms. This pair of primers is retained for the next primer combination detection.

[0056] (2) The relationship between multiple primers is relatively complex, and there is also a certain degree of competition and interference between primers. For the entire multiple primer system, in the initial system, this application adopted multiple alternative designs for each different type of gene when designing primers. After the primers were tested, the primers with poor amplification efficiency were eliminated and replaced, and repeated testing and adjustments were carried out.

[0057] Taking the 23S gene primer of the drug resistance gene of Mycoplasma genitalium (MG) as an example, MG_23S primer A is used to amplify the nucleic acid of Neisseria gonorrhoeae (NG). This primer has non-specific amplification of the nucleic acid of Neisseria gonorrhoeae (NG). The results are as follows: Figure 2 As shown in (A), primers need to be eliminated and replaced.

[0058] After primer optimization and adjustment, MG_23S primer B only specifically amplifies Mycoplasma genitalium (MG), but no longer non-specifically amplifies Neisseria gonorrhoeae (NG) nucleic acid. Figure 2 (B)

[0059] The specific sequence information of MG_23S primer A and MG_23S primer B before and after optimization is shown in Table 10.

[0060] Table 10 Specific sequence information of MG_23S primer A and MG_23S primer B

[0061] Since the detection position of drug-resistant genes is relatively fixed, the positions of certain primer sequences are relatively limited, and their Tm values ​​do not meet the required requirements. Preferably, the locked nucleotide design concept is introduced to keep the Tm values ​​of the primers as consistent as possible.

[0062] Taking the 23S drug-resistant gene primers of Mycoplasma genitalium as an example, the primers before and after optimization are shown in Table 11.

[0063] Table 11 Comparison of primers before and after optimization for the 23S drug resistance gene of Mycoplasma genitalium

[0064] Note: "+" indicates that the single base following the "+" is modified with locked nucleic acid.

[0065] The actual Tm value of the primer after synthesis is less than 55°C. The Tm value will be increased by 2-3 degrees after adding the locked nucleotide LAN design, which is compatible with the Tm value of other primer systems in multiple reactions.

[0066] Finally, 25 primer pairs amplified normally and the multiplex system was successfully constructed.

[0067] The specific primer sequences for the six STI infection pathogens are shown in Table 12.

[0068] Table 126 specific primer sequences for STI pathogens

[0069] Note: "+" indicates that the single base following the "+" is modified with locked nucleic acid.

[0070] (3) Optimization of primer concentration and reaction conditions During the amplification process using the above-mentioned primer sequences in equal proportions, the present applicant discovered that the actual amplification results were consistently below expectations. This was likely due to the relatively high or low amplification efficiency of some primers, which resulted in amplification bias in other sequences. Therefore, this example further optimized the primer concentrations within the primer system to improve the balance of amplification across the system, maximizing the sensitivity of detecting each identification or drug resistance gene in the sample, thereby ensuring the comprehensiveness and accuracy of subsequent nanopore sequencing.

[0071] The concentration of other identification / drug resistance primers in the system is 0.1 μM-0.2 μM. Preferably, the specific ratio of the identification gene and drug resistance gene primers is as follows: Primer01_R / F:Primer02_R / F:Primer03_R / F:Primer04_R / F:Primer05_R / F:Primer06_R / F:Primer07_R / F:Pr imer08_R / F:Primer09_R / F:Primer10_R / F:Primer11_R / F:Primer12_R / F:Primer13_R / F:Primer14_R / F:Prime r15_R / F: Primer16_R / F: Primer17_R / F: Primer18_R / F: Primer19_R / F: Primer20_R / F: Primer21_R / F: Primer22 _R / F: Primer23_R / F: Primer24_R / F: Primer25_R / F=3:2:2:4:4:4:4:3:3:3:4:4:4:4:2:2:3:3:4:2:2:3:4:4:3.

[0072] Through reaction condition testing and optimization, the reaction conditions of this application were established as follows: 94°C for 2 min; 94°C for 30 s, 60°C for 90 s, 34 cycles; 72°C for 10 min.

[0073] Prepare nucleic acid reference materials. Based on the digital PCR test results, quantify each microbial pathogen to 50,000 copies / mL. Then mix them in proportion, perform PCR amplification and library construction as described above, and finally perform sequencing on the machine. Using the gene sequence number as the reference standard, the number of drug-resistant gene sequences for each species should be relatively uniform, with no obvious amplification bias.

[0074] The results of PCR amplification and library construction using equal ratio mixing of primer sequences and adjusted ratio mixing are shown in Table 13.

[0075] Table 13 Comparison of sequencing results before and after primer sequence adjustment

[0076] The results showed that before adjustment, the primer sequences were mixed in equal proportions, and the target gene sequences were quite different. After the primer sequence mixing ratio was adjusted, the target gene primers amplified normally and evenly, the amplification efficiencies of each primer were close, and the multiplex system was successfully constructed.

[0077] Example 3 Sequencing Minimum Detection Limit Positive reference samples of known microorganism concentrations were prepared and serially diluted. The starting concentration of the reference sample was set at 3200 copies / mL, and the sample was diluted in a two-fold serial dilution to 1600 copies / mL, 800 copies / mL, 400 copies / mL, 200 copies / mL, 100 copies / mL, 50 copies / mL, and finally 25 copies / mL. Target samples were extracted, amplified, and sequenced according to the pre-defined concentration gradients. Each concentration was tested in parallel three times, and a negative control was included. The specific test results for each concentration gradient were recorded. The recorded data were analyzed to determine the minimum detectable concentration (LOD) for the sequencing platform: if the current dilution gradient tested positive, the previous dilution concentration tested positive, and the next dilution concentration tested negative, the current dilution gradient was determined to be the critical positive concentration.

[0078] Finally, the LOD values ​​of the detection performance of each pathogen of the kit were obtained: Identification genes: Ureaplasma urealyticum 50 copies / mL, Mycoplasma hominis 50 copies / mL, Mycoplasma genitalium 50 copies / mL, Chlamydia trachomatis 50 copies / mL, Neisseria gonorrhoeae 30 copies / mL, Trichomonas vaginalis 50 copies / mL.

[0079] Drug-resistant genes: Ureaplasma urealyticum 100 copies / mL, Mycoplasma hominis 100 copies / mL, Mycoplasma genitalium 100 copies / mL, Chlamydia trachomatis 100 copies / mL, Neisseria gonorrhoeae 80 copies / mL, Trichomonas vaginalis 100 copies / mL.

[0080] Example 4 Clinical Sample Verification This example tested and verified 15 real clinical samples (10 vaginal swabs and 5 underwear secretions), and verified the effectiveness and accuracy of the method by comparing it with traditional mNGS detection results and fluorescent quantitative PCR.

[0081] The test comparison results are shown in Table 14.

[0082] Table 14 Comparison of detection results of different methods

[0083] Results showed that the detection results of the present invention's method were essentially consistent when compared with those of traditional mNGS and fluorescent quantitative PCR. The detection rate for pathogens and drug-resistance genes was 100%, and its targeted amplification sensitivity was superior to that of mNGS. Furthermore, the third-generation sequencing method employed in this invention has the advantage of long length, enabling accurate differentiation between the two biotypes of Ureaplasma microti and Ureaplasma urealyticum, helping clinicians better assess their clinical significance.

[0084] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.

Claims

1. A primer combination for identification of six reproductive tract pathogens and detection of drug-resistant genes, characterized in that: The primer combination includes: (1) The primer combination for detecting Chlamydia trachomatis and its drug-resistant genes includes four pairs of primers: Primer01_R / F-Primer04_R / F, and the nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.8; (2) The primer combination for the detection of Mycoplasma genitalium and its drug-resistant genes includes four pairs of primers: Primer05_R / F-Primer08_R / F, and the nucleotide sequences are shown in SEQ ID NO.9-SEQ ID NO.16; wherein, in the Primer08_F sequence, the base "C" at position 21 and the base "G" at position 22 are both locked nucleic acids, and in the Primer08_R sequence, the base "C" at position 21 and the base "G" at position 23 are both locked nucleic acids; (3) The primer combination for the detection of Ureaplasma urealyticum and its drug-resistant genes includes five pairs of primers: Primer09_R / F-Primer13_R / F, and the nucleotide sequences are shown in SEQ ID NO.17-SEQ ID NO.26; wherein, in the Primer13_F sequence, the base "G" at position 20 is a locked nucleic acid; (4) The primer combination for the detection of Mycoplasma hominis and its drug-resistant genes includes four pairs of primers: Primer14_R / F-Primer17_R / F, and the nucleotide sequences are shown in SEQ ID NO.27-SEQ ID NO.34; wherein, in the Primer14_R sequence, the 18th base "C" is a locked nucleic acid; in the Primer16_R sequence, the 19th base "C" is a locked nucleic acid; (5) The primer combination for detecting Neisseria gonorrhoeae and its drug-resistant genes includes five pairs of primers: Primer18_R / F-Primer22_R / F, and the nucleotide sequences are shown in SEQ ID NO.35-SEQ ID NO.44; (6) The primer combination for the detection of Trichomonas vaginalis and its drug-resistant genes includes three pairs of primers: Primer23_R / F and Primer25_R / F, and the nucleotide sequences are shown in SEQ ID NO.45-SEQ ID NO.

50.

2. The primer combination for identification of six reproductive tract pathogens and detection of drug-resistant genes according to claim 1, characterized in that: Resistance genes include: (1) Targeting the A2057G, A2058C, A2059G, and T2611C ​​sites of the macrolide resistance gene 23S of Chlamydia trachomatis; targeting the S83I and S83R sites of the quinolone resistance gene gyrA; and targeting the R83G site of the quinolone resistance gene ParC; (2) Targeting the A2058G, A2058C, A2058T, and A2059G sites of the macrolide resistance gene 23S of Mycoplasma genitalium; targeting the M95I and D99N sites of the quinolone resistance gene gyrA; and targeting the S83R, S83I, S83N, D87N, D87Y, D87H, and D87G sites of the quinolone resistance gene ParC; (3) Targeting the G2654T and T2245C sites of the macrolide resistance gene 23S of Ureaplasma urealyticum; targeting the Q104K and D112E sites of the quinolone resistance gene gyrA; targeting the S83L, E87Q, E87K, and D82N sites of the quinolone resistance gene ParC; targeting the C1192T, G966T, and G967T sites of the tetracycline resistance gene 16S for plasmid-mediated resistance of the tetracycline resistance gene tetM; (4) Targeting the A2058G, A2059G, and G2057A sites of the macrolide resistance gene 23S of Mycoplasma hominis; targeting the S153A and S153L sites of the quinolone resistance gene gyrA; and targeting the S91I site of the quinolone resistance gene ParC; (5) Targeting the A2059G and T2611G sites of the macrolide resistance gene 23S of Neisseria gonorrhoeae; targeting the S91F, D95Y, D95A, D95G, and D95N sites of the quinolone resistance gene gyrA; targeting the D86N, S88A, S88P, E91A, E91G, E91K, and E91Q sites of the quinolone resistance gene ParC; and targeting the D345 amino acid insertion, A501T, and G542S sites of the penicillin resistance gene PenA; (6) Targeting the Y71Stop site of the metronidazole resistance gene Ntr4 and the K80Stop site of Ntr6 of Trichomonas vaginalis.

3. The primer combination for identification of six reproductive tract pathogens and detection of drug-resistant genes according to claim 1, characterized in that: In the primer combination, the concentration ratio of each primer is: Primer01_R / F:Primer02_R / F:Primer03_R / F:Primer04_R / F:Primer05_R / F:Primer06_R / F:Primer07_R / F:Pr imer08_R / F:Primer09_R / F:Primer10_R / F:Primer11_R / F:Primer12_R / F:Primer13_R / F:Primer14_R / F:Prime r15_R / F: Primer16_R / F: Primer17_R / F: Primer18_R / F: Primer19_R / F: Primer20_R / F: Primer21_R / F: Primer22 _R / F: Primer23_R / F: Primer24_R / F: Primer25_R / F=3:2:2:4:4:4:4:3:3:3:4:4:4:4:2:2:3:3:4:2:2:3:4:4:

3.

4. Use of the primer combination according to any one of claims 1 to 3 in the preparation of a product for detecting the identification and drug resistance genes of six reproductive tract pathogens.

5. A kit for the identification and drug-resistant gene detection of six reproductive tract pathogens, characterized in that: The kit comprises the primer combination according to any one of claims 1 to 3.

6. The kit for identifying six reproductive tract pathogens and detecting drug-resistant genes according to claim 5, characterized in that: The kit also includes sample nucleic acid extraction reagents, PCR amplification reagents, and nanopore sequencing reagents.

7. A method for identifying six reproductive tract pathogens and detecting drug-resistant genes for non-diagnostic and non-therapeutic purposes, characterized in that: The method uses the kit according to any one of claims 5 to 6, comprising the following steps: S1, pre-treatment of the sample to be tested; S2. Extracting nucleic acid from the sample to be tested using a nucleic acid extraction reagent; S3, preparing a PCR reaction system using the extracted nucleic acid of the sample to be tested and a PCR amplification reagent, and performing multiplex PCR amplification; S4. After the amplified product is purified, the library is constructed using nanopore sequencing reagents; S5. Perform high-throughput sequencing on the constructed library.

8. The method according to claim 7, wherein: The final concentration of the primer set in the PCR reaction system is 0.1 μM to 0.5 μM; the final concentration of the sample DNA is greater than or equal to 0.2 ng / μL; and the final concentration of the multiplex reaction amplification enzyme is 0.05 U / μL.

9. The method according to claim 7, wherein: The amplification conditions of the PCR amplification were 94° C. for 2 min; 34 cycles of 94° C. for 30 s, 60° C. for 90 s, and 72° C. for 90 s; and 72° C. for 10 min, with a hold at 12° C.

10. The method according to claim 7, wherein: It also includes a data analysis step: after logging off the machine, the qualified sequence is compared with the set reference sequence to obtain the mutation status of the common drug-resistant mutation sites of the reproductive tract pathogen identification genes and drug-resistant genes in the sample.

Images