Reagent and kit for identifying respiratory pathogens and detecting drug resistance and toxicity of respiratory pathogens based on single molecule sequencing method and application of reagent and kit

Abstract

The invention relates to the technical field of molecular biological detection, and particularly provides a reagent for respiratory tract infection pathogen identification and drug resistance and virulence gene detection. Each primer in the plurality of primers comprises a first sequence as shown in SEQ ID NO: 1-165 and an optional second sequence for distinguishing sample sources. Target spots detected by the super-multiplex primer combination comprehensively cover pathogen directories (totally 353 types) of latest respiratory tract infection diagnosis guidelines at home and abroad, low-cost, high-sensitivity, high-specificity and high-flux detection is achieved, and clinical medication can be effectively and comprehensively guided.

Description

Technical Field

[0001] The present application relates to the field of molecular biological detection technology, and specifically to a reagent, a kit and its application for identifying respiratory pathogens and detecting their drug resistance and virulence based on single-molecule sequencing. Background Art

[0002] Respiratory infections, a major global public health challenge, encompass a wide range of diseases caused by bacteria, fungi, viruses, and other microorganisms. They occur worldwide and are particularly common among children, the elderly, and those with compromised immune systems. Furthermore, the increasing prevalence of drug resistance in pathogens has become a major challenge in the treatment of respiratory infections. Currently, the diagnosis, treatment, monitoring, and prevention of tuberculosis (TB) rely on pathogen identification and multidrug susceptibility testing. Commonly used clinical methods include culture of Mycobacterium tuberculosis (M. tuberculosis), acid-fast smear microscopy, molecular biology, immunology, and imaging. Among these common methods, culture is the gold standard for TB diagnosis. However, its long culture cycle of 2-8 weeks makes it unsuitable for rapid diagnostic results and may even delay optimal treatment. Furthermore, conventional laboratories, due to their low biosafety levels, are unable to perform M. tuberculosis culture, severely limiting the practical application and efficiency of culture. Furthermore, although microscopic detection of Mycobacterium tuberculosis in sputum or other body fluids is a faster and simpler method, it has poor sensitivity and cannot predict drug resistance. Immunological techniques, including the interferon-gamma release assay (IGRA), measure the level of interferon-gamma released specifically for Mycobacterium tuberculosis, but are expensive and can cross-react with nontuberculous mycobacteria, affecting test accuracy.

[0003] Molecular diagnostic methods, including real-time PCR and sequencing, have emerged. Nucleic acid amplification testing (NAAT) is an important reference standard for the diagnosis of respiratory pathogens. Emerging rapid pathogen detection technologies, such as metagenomic sequencing (mNGS), single-molecule sequencing (TGS), and targeted sequencing (TS), also offer numerous options for respiratory pathogen detection. However, while NAATs (such as GeneXpert® MTB / RIF) can detect Mycobacterium tuberculosis and rifampicin resistance in sputum within 2 hours, like real-time PCR, they have a limited number of targets, making it difficult to comprehensively analyze the species and drug resistance of multiple respiratory pathogens. In clinical practice, metagenomic sequencing (including mNGS and mTGS) still faces three technical bottlenecks when applied to respiratory pathogen detection: first, the proportion of human host nucleic acids in clinical respiratory samples is too high (often reaching over 95%), resulting in dilution of the microbial nucleic acid load. Even with the use of technologies such as targeted capture, the sensitivity of pathogen detection is low. Second, the microbial composition of respiratory specimens is complex, making it difficult to distinguish between colonizing bacteria and pathogenic bacteria. For example, the colonization rate of Streptococcus pneumoniae in the nasopharynx of healthy people can reach 30%, and its genome coverage shows a nonlinear dynamic relationship with its pathogenic state. Therefore, the identification of pathogens often requires a combination of semi-quantitative thresholds and comprehensive analysis of clinical characteristics. Even more troublesome is that the current metagenomic sequencing (including mNGS and mTGS) detection technology lacks recognized data interpretation standards, resulting in an unclear relationship between sequencing results and treatment. The interpretation of massive data is extremely complex (involving multiple links such as raw data quality control, sequence alignment, microbial species annotation, gene prediction, and functional annotation), which requires high computing resources and analysts.

[0004] Therefore, there is an urgent need to provide a detection reagent and method for respiratory pathogen identification and drug resistance that is fast, covers a wide range of drug-resistant mutations, is simple, easy to use, and low-cost, with high sensitivity, high accuracy / specificity. Summary of the Invention

[0005] The first aspect of the present application provides a reagent for identifying respiratory tract infection pathogens and detecting drug resistance and virulence, comprising: a collection of multiple primers, each of the multiple primers comprising a first sequence, the first sequences being respectively shown in SEQ ID NOs: 1-165, wherein the identification of respiratory tract infection pathogens and the detection of drug resistance and virulence are all performed in the same primer pool comprising the collection.

[0006] In some embodiments, the length of the amplicons of the plurality of primers ranges from 200 to 2100 bp, and the amplicons are suitable for single molecule sequencing.

[0007] In some embodiments, the amount of each primer in the set of the plurality of primers in the reagent is the same.

[0008] In some embodiments, each primer in the plurality of primers further comprises a second sequence, wherein the second sequence comprises a tag sequence for distinguishing the source of the sample.

[0009] The second embodiment of the present application further proposes a kit for identifying pathogens of respiratory tract infection and detecting drug resistance and virulence, comprising: the reagents as described in any embodiment of the first aspect of the present application and one or more of the following: DNA polymerase, reverse transcriptase, dNTP, reaction buffer, Mg 2+ and single-molecule library construction reagents, wherein the single-molecule library construction reagents include one or more of the following: a single-molecule sequencing adapter, an end repair reagent, a ligation reagent for the single-molecule sequencing adapter, a purification reagent, and a tag sequence.

[0010] An embodiment of the third aspect of the present application further proposes a use of a reagent as described in any embodiment of the first aspect of the present application or a kit as described in any embodiment of the second aspect of the present application in preparing a preparation for identifying respiratory tract infection pathogens and detecting drug resistance and virulence, the use comprising: a. mixing the reagent with a nucleic acid sample to be tested to prepare a multiplex PCR reaction solution; b. placing the multiplex PCR reaction solution in a thermal cycling program to obtain amplicons of the target region of the nucleic acid sample to be tested based on multiple primers; c. performing single-molecule library construction and sequencing on the amplicons to obtain sequencing data of each of the amplicons; and d. parsing the sequencing data to identify respiratory tract infection pathogens in the nucleic acid sample and detect drug resistance and virulence, wherein the nucleic acid sample to be tested is DNA or RNA.

[0011] In some embodiments, based on the nucleic acid sample to be tested being RNA, before step a, the method further comprises: e. performing reverse transcription on the nucleic acid sample to be tested.

[0012] In some embodiments, the thermal cycling program includes: a preliminary denaturation phase: 98°C, 2-5 min; a cycling phase: 98°C, 15 s; 62°C, 30 s; 58°C, 30 s, 72°C, 1-5 min, 25-40 cycles; and a supplementary extension phase: 72°C, 5-10 min.

[0013] In some embodiments, the working concentration of each of the multiple primers in the multiplex PCR reaction solution is 10 μM, the amount of the nucleic acid sample to be tested is 0.1-100 ng, and the detection limit of the respiratory tract infection pathogen in the nucleic acid sample to be tested is 100 copies / μL.

[0014] In some embodiments, the nucleic acid sample to be tested is single or multiple, wherein based on the fact that the nucleic acid sample to be tested is multiple, step a includes: mixing the reagent with each of the nucleic acid samples to be tested to prepare the multiplex PCR reaction solution, wherein the reagent includes a set of multiple primers, wherein the set of primers includes multiple primers, each of the primers includes a first sequence and a second sequence, and the first sequence is shown as SEQ ID NO: 1-165 respectively; the second sequence includes a tag sequence for distinguishing the source of the sample.

[0015] In a fourth aspect, an embodiment of the present application further provides an integrated system for identifying respiratory tract infection pathogens and detecting drug resistance and virulence in multiple nucleic acid samples based on single-molecule sequencing, the system comprising: i. an amplification-preliminary library module, configured to amplify target regions associated with identification of respiratory tract infection pathogens and detection of drug resistance and virulence in the nucleic acid sample to be tested using a reagent as described in any embodiment of the first aspect of the present application to obtain amplicons of each target region, wherein the reagent comprises a set of multiple primers, wherein the set of primers comprises multiple primers, each of the primers comprises a first sequence and a second sequence, wherein the first sequences are respectively represented by SEQ ID NOs: 1-165, and the second sequence comprises a tag sequence for distinguishing the source of the sample; ii. a single-molecule sequencing adapter introduction module, configured to introduce a single-molecule sequencing adapter into one side of each amplicon to obtain a single-molecule sequencing library of the nucleic acid sample to be tested; iii. a single-molecule sequencing module, configured to perform single-molecule sequencing on the single-molecule sequencing library to obtain sequencing data for each amplicon; and iv. The data analysis module is used to analyze the sequencing data to identify the respiratory tract infection pathogens in the nucleic acid sample and detect drug resistance and virulence.

[0016] The technical solution of this application achieves the following technical effects: 1. This embodiment of the present application eliminates the need for microbiological culture of collected clinical respiratory samples. Instead, a multi-target primer combination amplifies the clinical sample nucleic acid directly, resulting in target fragments that are compatible with the long amplicons of a single-molecule long-read sequencer. Compared to traditional detection methods, the primer combination proposed in this application covers a wider range of common respiratory pathogens and their clinically relevant drug resistance and virulence genes, as well as species-specific 16S targets. The primer combination covers pathogens listed in the latest domestic and international respiratory infection diagnostic guidelines, as well as emerging pathogens not covered by existing technologies. Leveraging single-molecule nanopore long-read sequencing, the primer combination covers the V1-V9 regions of the 16S rDNA. This full-length sequence detection achieves extremely high species-level annotation and excellent bacterial taxonomy and species identification capabilities (traditional short-read sequencing only covers the V1-V2, V1-V3, V4, and V3-V5 regions of the 16S rDNA). The primer combination proposed in the examples of this application and the detection method using the same have a detection concentration as low as 1 ng / μL and 100 copies / μL of sample nucleic acid, with a sensitivity of 100% and a specificity of 100%, achieving a significant improvement in the sensitivity and accuracy of pathogen detection, and at the same time being able to provide effective guidance for clinical drug use.

[0017] 2. The embodiment of the present application realizes a streamlined number of primers and their coordination with each other through the special design of the primer combination. Compared with the primer design of the traditional second-generation sequencing platform, it optimizes the primer Tm value, GC content and secondary structure in multiple dimensions while ensuring the specificity of primer capture. The designed primer combination has no cross-reaction and extremely low mismatch rate when conducting comprehensive detection of common respiratory pathogens, showing high and balanced amplification efficiency and extremely high detection efficiency, especially in terms of sensitivity and specificity. At the same time, the special design of the multiple primer combination also greatly reduces the demand for templates and detection reagents, and can save about 2 hours of label sequence connection time in traditional library construction, thereby greatly shortening the detection time while reducing the detection cost.

[0018] 3. The multiplex PCR detection method proposed in the embodiment of the present application can also realize the simultaneous detection of multiple samples. By combining the detection method with the drug resistance result analysis software, an integrated "detection-analysis-reporting" system can be formed. Compared with traditional detection and analysis, the present application shortens the turnaround time of the entire process from the original nearly 8 hours to about 5 hours, shortening the cycle by about 37.5%; at the same time, the present application can realize the simultaneous detection of multiple samples while reducing the experimental time and steps, with faster detection speed, less chip consumption, high sensitivity and strong specificity. The integrated system can achieve high-throughput, hourly "from sample to report" fast, efficient and high-accuracy detection.

[0019] 4. The embodiments of the present application can also be equipped with technical analysis software designed specifically for single-molecule sequencing data of each amplicon. For clinical samples, generally only 600 Mb of sequencing data is required to achieve respiratory pathogen identification and rapid and accurate prediction of drug resistance and virulence genes through such technical analysis software. For example, equipped with a 64-bit central processing unit, the entire process analysis can be completed within 10 minutes, thereby greatly reducing the data volume required in traditional analysis and the demand for computing power, making detection faster, more convenient and economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 Schematic diagram of the primer structure according to the embodiment of the present application; Figure 2 The following is an analysis process for sample single-molecule sequencing data according to an embodiment of the present application. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0023] This application creatively designs primer combinations for the identification of respiratory tract infection pathogens and the one-time detection of multiple drug resistance genes and virulence genes, an integrated library construction solution for simultaneous detection of multiple samples, and a complete data analysis process for this solution, providing a fast, stable, highly sensitive, highly accurate and low-cost feasible solution that guides the entire process from sample to report.

[0024] The first embodiment of the present application proposes a reagent for identifying respiratory tract infection pathogens and detecting drug resistance and virulence, comprising: a set of multiple primers, each of the multiple primers comprising a first sequence, the first sequences being respectively shown as SEQ ID NOs: 1-165 in Table 1, wherein the identification of respiratory tract infection pathogens and the detection of drug resistance and virulence are all carried out in the same primer pool comprising the set.

[0025] Table 1

[0026] The targets detected by the primer combinations proposed in the examples of this application fully cover the pathogen catalog of the latest domestic and international respiratory infection diagnostic guidelines, covering a total of 353 species including bacteria, fungi, DNA viruses, RNA viruses, parasites, mycoplasmas, chlamydia, ureaplasmas, spirochetes, and rickettsiae (see Table 2). This covers the pathogen spectrum epidemiological data reported in core journals in the field of respiratory infections over the past decade. Therefore, the primer combinations provided by this application can achieve effective and comprehensive identification of respiratory infection pathogens and guide clinical drug use. In addition, the special design of the primer combination in this application achieves a streamlined number of primers and their mutual synergy. Compared with the primer design of traditional second-generation sequencing platforms, it has multi-dimensionally optimized Tm values, GC content, and secondary structure while ensuring primer capture specificity. It can achieve simultaneous and stable amplification of each target in ultra-multiplex PCR with 165 primers simultaneously, without cross-reaction and with extremely low mismatch rate, showing high and balanced amplification efficiency and extremely high detection efficiency, especially in terms of sensitivity and specificity. The primer combination proposed in the embodiment of the present application is designed for a single-molecule long-read sequencing platform, and can fully cover the V1-V9 region of the pathogen's 16s rDNA. Compared with traditional short-read sequencing that only covers the V1-V2, V1-V3, V4, and V3-V5 regions of the 16s rDNA, the primer combination proposed in the embodiment of the present application achieves an extremely high level of annotation at the species level and excellent bacterial classification and species identification capabilities. Based on its special design and combination with a single-molecule platform, the primer combination proposed in the embodiment of the present application can achieve rapid, accurate, and high-precision identification of respiratory tract infection pathogens and their comprehensive drug resistance and virulence gene one-time detection, with a detection concentration as low as 1 ng / μL and 100 copies / μL sample nucleic acid, a sensitivity of 100%, and a specificity of 100%, achieving a significant improvement in the sensitivity and accuracy of pathogen detection, and at the same time being able to effectively and comprehensively guide clinical drug use.

[0027] Table 2: Classification of 353 pathogens

[0028] Table 3: Details of 353 pathogens

[0029] In some embodiments, the length of the amplicon of the multiple primers ranges from 200 to 2100 bp, for example, 200-400, 300-800, 400-1000, 500-2000 bp or any value or range therebetween, and these long amplicons are suitable for single molecule sequencing. In some embodiments, single molecule sequencing includes nanopore sequencing. In the embodiments of the present application, each primer is designed to amplify a long amplicon adapted for a single molecule long read length sequencer, thereby completely covering the V1-V9 region of 16s rDNA, and can be combined with single molecule sequencing to perform real-time reading of the entire sequence of a single molecule, thereby achieving high-throughput, hourly "from sample to report" fast, efficient, and highly accurate pathogen identification and detection of drug resistance and virulence genes.

[0030] In some embodiments, in addition to the first sequence shown in SEQ ID NO: 1-165, each primer in the plurality of primers further comprises a second sequence, wherein the second sequence comprises a tag sequence (such as a barcode) for distinguishing the source of the sample. In some embodiments, the tag sequence can be located on the 5' side of the first sequence (such as Figure 1 As shown). It is understandable that the second sequence (label sequence) carried by the primers used to amplify the same sample is the same; the second sequence (label sequence) carried by the primers used to amplify different samples is different. In the embodiment of the present application, by introducing the label sequence into each primer respectively, the introduction of labels for multi-sample detection in the conventional single-molecule sequencing library construction process can be avoided, the operation time of the label introduction step (about 2 h) is saved, and the "amplification-label labeling (i.e., library pre-preparation)" is achieved in one step, thereby greatly simplifying the experimental operation, shortening the detection time, and improving the detection throughput. It is understandable that in addition to the label sequence, the second sequence may also contain one or more other sequences, such as universal sequences (i.e., fixed sequences, etc.) used for enrichment and purification, sequencing adapter identification / connection, etc. The present application does not limit the types of other sequences contained in the second sequence.

[0031] In some embodiments, the primers in the set of multiple primers have the same content. In some embodiments, the working concentration of each of the multiple primers is 10 μM or less. The primer combinations proposed in the embodiments of the present application, after being mixed at equal concentrations, can achieve synchronous and stable amplification of each target under super-multiplex PCR with 165 primers participating simultaneously, without cross-reaction and with extremely low mismatch rate, showing high and balanced amplification efficiency and extremely high detection efficiency, especially in terms of sensitivity and specificity.

[0032] In some embodiments, the reagents for the identification of respiratory tract infection pathogens and the detection of drug resistance and virulence genes can also introduce primers for other target genes or target mutation sites according to specific needs, such as primers for other targets used to identify respiratory tract infection pathogens, such as 16S rRNA, IS6110 , 16S-23S rRNA gene spacer (ITS), etc.; as well as other drug resistance or virulence target genes, etc., all of which fall within the scope of protection of this application.

[0033] The second embodiment of the present application provides a kit for identifying pathogens of respiratory tract infection and detecting drug resistance and virulence, comprising: the reagents as described in any embodiment of the first aspect of the present application and one or more of the following: DNA polymerase, reverse transcriptase, dNTP, reaction buffer, Mg 2+ And single-molecule library building reagents, wherein the single-molecule library building reagents include one or more of the following: single-molecule sequencing adapters, end repair reagents, connection reagents for the single-molecule sequencing adapters, and purification reagents. In some embodiments, based on the fact that each primer does not contain a second sequence containing a label sequence, the single-molecule library building reagent may also include a label sequence for distinguishing different samples. In some specific embodiments, the working concentration of each primer in the primer set in the reagent is 10 μM, wherein each primer is provided in the form of equal concentration. It is understandable that in addition to the above reagents, the kit may also include sequencing reagents for single-molecule sequencing, etc., all of which fall within the scope of protection of this application.

[0034] The third embodiment of the present application proposes a method for identifying respiratory tract infection pathogens and detecting drug resistance and virulence, comprising: a. mixing the reagents described in any embodiment of the first aspect of the present application with the nucleic acid sample to be tested to prepare a multiplex PCR reaction solution; b. placing the multiplex PCR reaction solution in a thermal cycle program to obtain amplicons of each target region of the nucleic acid sample to be tested based on multiple primers; c. performing single-molecule library construction and sequencing on the amplicons to obtain sequencing data of each amplicon; and d. parsing the sequencing data to identify respiratory tract infection pathogens in the nucleic acid sample and detect drug resistance and virulence.

[0035] In some embodiments, the nucleic acid sample to be tested is DNA or RNA. Based on the nucleic acid sample to be tested being RNA, before step a, the method further comprises: e. performing reverse transcription on the nucleic acid sample to be tested.

[0036] In some embodiments, the working concentration of each primer in the primer set in the reagent is 10 μM, wherein each primer is provided in equal concentrations.

[0037] In some embodiments, the amount of nucleic acid sample to be tested is 0.1-300 ng, for example, 0.1-100, 0.1-150, 0.1-200 ng, or any value or range therebetween. In some embodiments, taking a 50 μL system as an example, the multiplex PCR reaction solution may include: a. Reaction mixture, including DNA polymerase, dNTP, Mg 2+The following preparations are prepared: a. a. primer mix, which is a mixture of equal amounts of each primer: 10 μL each, 10 μM each; c. a. test nucleic acid sample: 1 μL, at a concentration of 10-250 ng / μL; and d. sterile water: bring to a total volume of 50 μL. The primer combinations proposed in the examples of this application enable highly specific and accurate pathogen identification and detection of drug resistance and virulence genes with extremely low sample volumes. In some embodiments, the limit of detection of respiratory pathogens in the test nucleic acid sample is as low as 100 copies / μL.

[0038] In some embodiments, the thermal cycling program includes: a preliminary denaturation phase: 98°C, 2-5 min; a cycling phase: 98°C, 15 s; 62°C, 30 s; 58°C, 30 s; 72°C, 1-10 min, preferably 1-5 min, more preferably 110 s, 25-40 cycles, preferably 30-35 cycles; a supplementary extension phase: 72°C, 5-10 min; and an optional storage phase: 4°C-10°C.

[0039] In the embodiments of the present application, the ratio of each component in the amplification system, primer concentration, etc. are adaptively optimized based on the primer combination, thereby reducing cross-reactions between primers and solving compatibility issues in traditional multiplex PCR (such as primer dimers, differences in amplification efficiency, etc.); at the same time, by adaptively adjusting the amplification program, optimized reaction parameters are provided to ensure that multiple targets can be amplified simultaneously in the same reaction system, thereby providing a basis for high-throughput, low-cost, high-sensitivity, and high-accuracy identification of respiratory pathogen species and detection of drug resistance and toxicity.

[0040] In some embodiments, the nucleic acid sample to be tested may be single or multiple, wherein based on the number of nucleic acid samples to be tested, step a may include: mixing the reagents with each nucleic acid sample to be tested to prepare a multiplex PCR reaction solution, wherein the reagents include a set of multiple primers, wherein the set of primers includes multiple primers, each primer includes a first sequence and a second sequence, the first sequence is shown as SEQ ID NO: 1-165 respectively; the second sequence includes a label sequence for distinguishing the source of the sample. That is, the embodiment of the present application proposes a method for identifying respiratory tract infection pathogens and detecting drug resistance and virulence for multiple samples, wherein the special design of the label sequence of each primer can realize the integrated amplification and library construction of multiple samples, which saves about 2 hours of connection time in traditional library construction; at the same time, the demand for templates and detection reagents by each primer is also extremely low, thereby greatly shortening the detection time and reducing the detection cost.

[0041] In some embodiments, as Figure 2As shown, step d: parsing the sequencing data to identify respiratory tract infection pathogens in the nucleic acid sample and detect drug resistance and virulence may include the following steps: i. Input long-read targeted amplification data and filter the data using tools such as NanoFilt. ii. Create your own alignment database based on the target sequence corresponding to the amplicon. Commonly used databases include the bacterial 16S database (UNITE, https: / / unite.ut.ee / ; FROGS, https: / / frogs.toulouse.inra.fr / ), the rpoB database (FROGS, https: / / frogs.toulouse.inra.fr / ), the fungal ITS database (silva, https: / / www.arb-silva.de / ; UNITE, https: / / unite.ut.ee / ; FROGS, https: / / frogs.toulouse.inra.fr / ), the viral database (GenBank, https: / / www.ncbi.nlm.nih.gov / genbank / ), the bacterial drug resistance database (ResFinder, http: / / genepi.food.dtu.dk / resfinder), and the bacterial virulence database (VFDB, https: / / www.mgc.ac.cn / VFs / ). iii. Use a long-read alignment tool such as Minimap2 to align the filtered high-quality sequences to the reference sequence or reference genome of each target to obtain target coverage and the number of alignments, and calculate the coverage depth.

[0042] The above workflow has been developed using Python 3.10.6 as the software pathogen_detection.py (Software Copyright No. 2024SR1731913), which is available for direct download. This tool automates targeted sequencing data processing, including adapter removal, data quality control, long-read alignment, and statistical analysis. This tool provides efficient and accurate identification of respiratory pathogens and comprehensive detection of multi-drug resistance and virulence genes. The command is: python pathogen_detection.py input_path qlt database output_dir (where q specifies the NanoFilt minimum quality threshold; sequences below this threshold are filtered out; a value of 7 is recommended; l specifies the NanoFilt minimum length threshold; sequences shorter than this threshold are filtered out; a value of 0 is recommended; t specifies the number of Minimap2 alignment threads; recommended values are 12 and 24).

[0043] The method proposed in the embodiment of the present application is aimed at the demand of clinical multi-sample synchronous detection, and provides a technical process for multi-sample integrated library construction and a database and a complete analysis scheme for analyzing the output data. Compared with the traditional single-molecule nanopore sequencing library construction method, the multi-sample integrated library construction can shorten the library construction time by about 2 hours, and can realize the synchronous detection of multiple samples while reducing the experimental time and steps, with faster detection speed, less chip consumption, high sensitivity and strong specificity. In addition, using the deployment of independently developed databases and systematic tools, the analysis tool can run efficiently under lower computing resources-for clinical samples, only 600 Mb of sequencing data is needed to realize respiratory pathogen identification and accurate prediction of drug resistance and virulence genes through this process. For a 64-bit central processing unit, the entire data analysis process can be completed within 10 minutes. Thus, the method proposed in the embodiment of the present application not only greatly shortens the detection time, improves the detection speed, and reduces the data volume demand, but also reduces the demand for high-end computing equipment, making detection faster, more convenient and economical.

[0044] The fourth aspect embodiment of the present application proposes the use of the reagent as described in any embodiment of the first aspect of the present application or the kit as described in any embodiment of the second aspect of the present application in the preparation of preparations for the identification of respiratory tract infection pathogens and the detection of drug resistance and virulence.

[0045] In a fifth aspect, an embodiment of the present application provides an integrated system for identifying respiratory tract infection pathogens and detecting drug resistance and virulence in multiple nucleic acid samples based on single-molecule sequencing, comprising: i. an amplification-preliminary library module for amplifying target regions related to identification of respiratory tract infection pathogens and detecting drug resistance and virulence in the nucleic acid sample to be tested using reagents as described in any embodiment of the first aspect of the present application to obtain amplicons of each target region, wherein the reagents include a set of multiple primers, wherein the set of primers includes multiple primers, each of the primers includes a first sequence and a second sequence, wherein the first sequences are respectively represented by SEQ ID NOs: 1-165, and the second sequence includes a tag sequence for distinguishing the source of the sample; ii. a single-molecule sequencing adapter introduction module for introducing a single-molecule sequencing adapter into one side of each amplicon to obtain a single-molecule sequencing library of the nucleic acid sample to be tested; iii. a single-molecule sequencing module for performing single-molecule sequencing on the single-molecule sequencing library to obtain sequencing data for each amplicon; and iv. The data analysis module is used to analyze the sequencing data to identify the respiratory tract infection pathogens in the nucleic acid sample and detect drug resistance and virulence.

[0046] In the examples of this application, any or all of the above-mentioned quality control filtering, alignment, and drug resistance and virulence gene prediction steps can be combined to form a more efficient automated script. Furthermore, it is understood that other command lines, scripts, or software can also be used to analyze sequencing data, and this application does not limit the specific analysis script used.

[0047] It should be noted that the explanation of the embodiment of "reagents for identification of respiratory tract infection pathogens and detection of drug resistance and virulence" in this application is also applicable to the kit containing the reagent, the method and system using the same, which also have the same technical effects and advantages, and will not be repeated in this application.

[0048] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0049] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0050] Except for the special parameters indicated, the data analysis in the following examples all refers to conventional procedures in the art, such as using conventional analysis methods and / or default parameters of relevant software.

[0051] Example 1 In this example, primer combinations as shown in SEQ ID NOs: 1-165 were used to PCR amplify ZYMO D6311 bacterial and fungal DNA standards (ZymoBIOMICS Microbiome Standard ((species distribution is not uniform), 220 ng, 20 μL, 11 ng / μL) and its ten-fold dilution (D6311 1:10 , 1.1 ng / μL, D6311 1:100 , 0.11 ng / μL and D6311 1:1000 , 0.011 ng / μL) to detect bacteria and fungi to verify the feasibility of the primer combination and detection method proposed in the examples of this application. The specific steps are as follows: 1.1 Multiplex PCR 1.1.1 The primer combinations shown in SEQ ID NOs: 1-165 were mixed in equal amounts to prepare a 10 μM primer mixture. Multiplex PCR reaction solutions were prepared according to the system shown in Table 4 to amplify ZYMO D6311 bacterial and fungal DNA standards (11 ng / μL), 1:10 diluted D6311, and 10 μM primer mixtures. 1:10 (1.1 ng / μL), D6311 diluted 1:100 1:100 (0.11 ng / μL), D6311 diluted 1:1000 1:1000 (0.011 ng / μL): Table 4

[0052] 1.1.2 Place the prepared multiplex PCR reaction solution in a PCR instrument and perform amplification using the following thermal cycle program: (1) Pre-denaturation: 98°C, 3 min; (2) Denaturation, annealing, and extension: 98°C, 15 s, 62°C, 30 s, 58°C, 30 s, 72°C, 110 s, 35 cycles; (3) Supplementary extension: 72°C, 5 min; (4) Cool down to 4°C and hold.

[0053] 1.1.3 Purify the reaction product using magnetic beads and perform quality control on the purified reaction product.

[0054] 1.2 Single-molecule long-read library construction for a single sample Using the H940-000013 CycloneSEQ Universal Library Prep set (24 RXN), the purified reaction products were subjected to single-molecule library construction, strictly following the included instructions. This included a barcode attachment step, which took approximately 2 hours, to distinguish between different sample sources.

[0055] 1.3 Single-molecule long-read sequencing Use the H940-000016 CycloneSEQ WT Sequencing Kit (6T) and strictly follow its instructions to perform single-molecule long-read sequencing on the single-molecule library in 1.2.

[0056] 1.4 Data Analysis Refer to Figure 2 The analysis process shown uses the software pathogen_detection.py (Software Copyright Registration Number: 2024SR1731913) to perform statistics and analysis on the offline data.

[0057] 1.5 Results Table 5 shows the concentration of the amplified product in step 1.1.3 and the single-molecule long-read sequencing data results in step 1.4. As can be seen from Table 5, D6311 1:100 (0.11 ng / μL) and D6311 1:1000 The concentration of the amplified product (0.011 ng / μL) was low and did not meet the library construction requirements, so no subsequent single-molecule library construction was performed on it. At the same time, the data statistics in Table 5 show that the standard stock solution ZYMO D6311 (11 ng / μL) and D6311 1:10 (1.1 ng / μL) achieved effective amplification, and the N50 length (the length of a sequence when the sum of sequences arranged from longest to shortest reaches 50% of the total length) met the designed amplicon length, demonstrating that the primer combination designed in this example can amplify all targets. The sequencing data volume also meets the analysis requirements, indicating that the primer combination proposed in this example achieves efficient amplification. Furthermore, this result suggests that the detection limit of this primer combination is as low as 1.1 ng / μL (basically consistent with the requirements of the rTaq Multiplex 2X Master Mix for sample nucleic acids).

[0058] The target genes in the ZYMO D6311 bacterial and fungal DNA standards and their ten-fold dilution samples used in this example mainly include bacterial 16s rDNA genes, rpoBGene and fungal ITS regions. The species included in the standard include: eight bacteria: (1) Listeria monocytogenes, (2) Pseudomonas aeruginosa, (3) Bacillus subtilis, (4) Salmonella enterica, (5) Escherichia coli, (6) Lactobacillus fermentum, (7) Enterococcus faecalis, and (8) Staphylococcus aureus; and two fungi: (9) Saccharomyces cerevisiae and (10) Cryptococcus neoformans.

[0059] Table 6 shows the primer combination pair of ZYMO D6311 (11 ng / μL) and D6311 1:10 As shown in Table 6, the detection of each target in ZYMO D6311 (11 ng / μL) and its ten-fold diluted sample D6311 1:10 The detection data volume (1.1 ng / μL) is sufficient to meet analytical requirements. Based on this data, bacterial and fungal species can be well distinguished, achieving accurate identification of each pathogen. This result also indirectly demonstrates the effectiveness of the super-multiplex PCR primer combination and the corresponding reaction system and reaction procedure used in this example, and proves the technical reliability of the entire process from sample to report.

[0060] Table 5

[0061] Table 6

[0062] The experimental results above demonstrate that the primer combination in this example exhibits excellent amplification performance when detecting mixed multispecies (bacterial and fungal DNA standards with uneven distribution). Each primer accurately and sensitively captures the specific target of the target species (with a detection limit as low as 1.1 ng / μL), laying a solid foundation for subsequent sequencing and analysis. Furthermore, sequencing validation demonstrates that the primer combination and process design in this example can be applied to a single-molecule nanopore long-read platform, effectively distinguishing between different microbial species and specific targets, providing strong technical support for in-depth research on microbial communities.

[0063] Example 2 In this example, primer combinations containing sequences shown as SEQ ID NOs: 1-165 (i.e., first sequences) and sample tag sequences (i.e., second sequences, randomly selected from the linker barcodes LB01-LB24 in the H940-000018 CycloneSEQ 24 Barcode Library Prep Set (4 RXN), in this example, using tag #2 LB02 and tag #6 LB06, respectively) were used to detect bacterial and fungal targets in the ZYMO D6311 bacterial and fungal DNA standard (ZymoBIOMICS Microbiome Standard ((species non-uniform distribution), 220 ng, 20 μL, 11 ng / μL), and a multi-sample library construction and sequencing process was established. The specific steps are as follows: 2.1 Multiplex PCR Referring to the method in step 1.1 of Example 1, multiple standard samples were amplified using primer combinations comprising the above-mentioned first sequence and second sequence, wherein the second sequence of the primer combinations used in different samples was different, i.e., tag #2 and tag #6.

[0064] 2.2 Single-molecule long-read library construction for multiple samples The purified reaction products were used to construct a single-molecule library using the H940-000013 CycloneSEQ Universal Library Prep set (24 RXN) in strict accordance with its instructions, omitting the sample barcode connection step.

[0065] 2.3 Single-molecule long-read sequencing Refer to step 1.3 of Example 1.

[0066] 2.4 Data Analysis Refer to step 1.4 of Example 1.

[0067] 2.5 Results Sample label #2 and sample label #6 were randomly selected to mark the sequencing samples of two groups of amplicons of ZYMO D6311, respectively. Table 7 shows the concentration of the amplified products of the nucleic acid samples and the statistics of the single-molecule long-read sequencing data of the primer combination with the sample label sequence of the present application. As can be seen from Table 7, the primer combination with the sample label sequence also performed stably when amplifying the target region, and both achieved effective amplification of the target region, indicating that the primer combination designed in this embodiment can be used to synchronously amplify the various targets of multiple samples, and the amount of sequencing data meets the analysis requirements, indicating that the primer combination proposed in this embodiment achieves efficient amplification.

[0068] Table 8 shows the detection of each target in ZYMO D6311 by the primer combination of this example. As can be seen from Table 8, under the guidance of the primer combination with the sample tag sequence, the detection data volume of ZYMO D6311 is sufficient to meet the analysis requirements. Based on these data, various bacterial and fungal species can be well distinguished, and accurate identification of various pathogens can be achieved. This result also indirectly reflects the effectiveness of the super-multiplex PCR primer combination in this example and the corresponding reaction system and reaction procedure, and proves the technical reliability of the multi-sample integrated detection process from sample to report.

[0069] Table 7

[0070] Table 8

[0071] The above experimental results show that the primer combination proposed in this example exhibits excellent amplification performance by detecting mixed multi-species (non-uniformly distributed bacteria and fungi) DNA standards. Each primer can accurately and sensitively capture the specific target of the target species, laying a solid foundation for subsequent sequencing and analysis. In addition, sequencing verification proves that the primer combination and process design of this example can be applied to the single-molecule nanopore long-read platform, and can effectively distinguish different microbial species and specific targets, providing strong technical support for in-depth research on microbial communities. At the same time, using the primer combination of this example for amplification omits the two-hour tag connection step in traditional library construction, thereby achieving a high level of adaptation of the multi-sample integrated library construction system and the single-molecule nanopore long-read sequencing platform, and has faster detection speed, lower chip consumption, high sensitivity, and strong specificity.

[0072] Example 3 In this example, primer combinations containing sequences shown in SEQ ID NOs: 1-165 (i.e., first sequences) and sample tag sequences (i.e., second sequences, randomly selected from the concatenated barcodes LB01-LB24 in the H940-000075 CycloneSEQ 24 Barcode Library Prep Set (4 RXN)—in this example, tag #1 LB01 and tag #2 LB02, respectively) were used to test the capture and analysis of target regions in mixed samples containing bacteria and fungi using the multi-sample integrated analysis process established in Example 2. This was done to evaluate the detectability of the primer combination proposed in this example for multi-species samples. The mixed bacterial and fungal samples used were ATCC MSA-4000 bacterial DNA standards (with a non-uniform distribution of species, approximately 1 ng / μL) and ATCC MSA-1010 fungal DNA standards, corresponding to 12 target sequences and 10 fungal targets, respectively. Both samples comprehensively cover bacterial and fungal species, as well as bacterial resistance genes. Specific steps are similar to those in Example 2. The sequencing results of two groups of sequencing tags #1 and #2 were randomly selected for display, and the results are shown in Tables 9 and 10.

[0073] Table 9 shows the concentrations of amplified products for two sample groups, sequencing tag #1 and #2, as well as single-molecule long-read sequencing data statistics. As shown in Table 9, the primer combination proposed in this example guides high concentrations of amplified products for each sample. Under the integrated library construction process incorporating tag sequences, the constructed libraries are of high quality, and sequencing generates sufficient data to fully meet analysis requirements. This demonstrates that the primer combination proposed in this example achieves efficient amplification based on the reaction system and reaction procedure of this example, and performs well in the integrated library construction process based on it.

[0074] Table 10 shows the detection of 22 target regions of mixed samples containing bacteria and fungi by the primer combination of this embodiment. As can be seen from Table 10, under the guidance of the primer combination with the sample tag sequence, the amplification and library construction sequencing process of this embodiment can effectively capture and analyze each target sequence in the sample, achieving a detection rate of 100%. Based on these data, the various bacterial species and fungal species can be well distinguished to achieve accurate identification of each pathogen. In addition, the sequencing results of the "multi-sample integrated library construction" method can also distinguish different samples well, reflecting the effectiveness of the super-multiplex PCR reaction system and the "multi-sample integrated library construction" method in this embodiment, and proving the technical reliability of the multi-sample integrated detection process from sample to report.

[0075] Table 9

[0076] Table 10

[0077] The above experimental results show that the primer combination proposed in this example exhibits excellent amplification performance by detecting mixed multi-species (non-uniformly distributed bacteria and fungi) DNA standards. Each primer can accurately and sensitively capture the specific target of the target species, laying a solid foundation for subsequent sequencing and analysis. In addition, sequencing verification proves that the primer combination and process design of this example can be applied to the single-molecule nanopore long-read platform, and can effectively distinguish different microbial species and specific targets, providing strong technical support for in-depth research on microbial communities. At the same time, using the primer combination of this example for amplification omits the two-hour tag connection step in traditional library construction, thereby achieving a high level of adaptation of the multi-sample integrated library construction system and the single-molecule nanopore long-read sequencing platform, and has faster detection speed, lower chip consumption, high sensitivity, and strong specificity.

[0078] Example 4 This example uses a primer combination containing the sequence shown in SEQ ID NO: 1-165 (i.e., the first sequence) and a sample tag sequence (i.e., the second sequence) to detect pathogens and drug resistance and virulence genes in clinical respiratory samples. The specific steps are as follows: 4.1 Multiplex PCR Referring to the method described in step 1.1 of Example 1, primer combinations containing the sequences shown in SEQ ID NOs: 1-165 (i.e., the first sequence) and the sample tag sequence (i.e., the second sequence) were used to amplify the following samples: sputum, bronchoalveolar lavage fluid, pleural effusion, and throat swab. All four samples were provided by Zhongshan Hospital Affiliated to Fudan University. The clinical microbiology laboratory detected predominantly nontuberculous mycobacteria. The bronchoalveolar lavage fluid sample was loaded into the reaction system at a volume of 1 μL, and its initial sample concentration is shown in Table 11.

[0079] 4.2-4.4 Single-molecule long-read library construction for multiple samples 4.5 Results Table 11 shows the original nucleic acid concentration, amplification product concentration, and subsequent sequencing data volume and analysis results for each sample in this example. As shown in Table 11, the primer combination proposed in this example can efficiently amplify each target sequence in the original sample, while producing sufficient data to meet subsequent analysis requirements. Furthermore, the positive species identified using the primer combination and multi-sample analysis process of this example are consistent with prior results, demonstrating that the primer combination and process of this example accurately identify each species in the sample. Furthermore, Table 11 shows that this example can effectively detect each pathogen with a sample concentration as low as 0.344 ng / μL (see reference 15), requiring an average data volume of only approximately 500 Mb. This demonstrates the high sensitivity, high accuracy, and extremely low computing power requirements of the single-molecule sequencing-based primer combination and process proposed in this example for clinical sample analysis.

[0080] Table 11

[0081] The above experimental results show that the primer combination proposed in the embodiment of the present application has good performance in species identification and drug resistance and virulence target amplification of respiratory pathogens in clinical samples. The primers can accurately, sensitively and specifically capture the designed various respiratory pathogen targets, thereby achieving targeted amplification of the targets to be tested, meeting the detection of various pathogens including viruses, bacteria, fungi, parasites and atypical pathogens in clinical samples, as well as the detection of key functional genes such as drug resistance genes. The sequencing results also show that the primer combination and detection method designed in this application can be used for the identification of various respiratory pathogens in clinical respiratory samples and the detection of their drug resistance genes and virulence genes. By using specially designed primers, the label (barcode) introduction step that takes about two hours in the multi-sample library construction process can be omitted, thereby greatly shortening the detection time. In addition, the primer combination and detection method proposed in the embodiments of the present application can be adapted to the multi-sample integrated library construction system and the single-molecule nanopore long-read sequencing platform. It has faster detection speed, less chip consumption, high sensitivity, strong specificity, and achieves high-accuracy detection (consistent with the clinical pathogen detection results).

[0082] Industrial Applicability This application creatively designs a super-multiple primer combination (including 165 primer sequences) for the identification of 353 species of respiratory infection pathogens and the detection of drug resistance and virulence genes, a multi-sample efficient library construction method based on it, and a sequencing data analysis process. By performing super-multiple PCR amplification of the target gene under a suitable reaction system and reaction procedure, constructing a single-molecule length long sequencing library for the amplicon, and performing single-molecule sequencing and data analysis on the library, accurate, sensitive, and comprehensive identification of respiratory infection pathogens and detection of their drug resistance and virulence genes are achieved. Through the special design of the primer combination and the detection process, the primer combination proposed in the embodiment of this application and the detection method based on it achieve low-cost, high-sensitivity, high-specificity, and high-throughput detection. At the same time, the detection cycle is short, the accuracy is high, and the genetic detection is comprehensive, fully covering the pathogen spectrum epidemiological data reported in the core journal literature in the field of respiratory infections in the past ten years, which can achieve effective and comprehensive identification of respiratory infection pathogens and guide clinical drug use.

[0083] In summary, compared to conventional pathogen detection solutions, the specially designed primer combination proposed in the embodiments of this application has the following core advantages: 1. Broader target coverage and higher clinical value: The primer combination proposed in this application example transcends the limitations of conventional pathogen detection by integrating five major pathogen categories associated with respiratory tract infections (including viruses, bacteria, fungi, parasites, and atypical pathogens) and key functional genes (such as drug resistance genes), enhancing pathogen identification and infection mechanism analysis capabilities in multiple dimensions. This not only reduces missed detection rates but also guides precise clinical medication decisions.

[0084] 2. Streamlined design and more stable performance: Compared with primer design solutions based on next-generation sequencing (NGS), this solution uses fewer primers, which effectively reduces the risk of primer dimer formation and nonspecific amplification, improves reaction efficiency, reduces reagent consumption, and lowers the cost per test.

[0085] 3. Highly compatible with long-read sequencing platforms, enabling rapid and accurate detection: A selection of 165 specially designed primers with optimized amplicon lengths are perfectly suited for single-molecule long-read sequencing platforms. This solution enables a rapid sample-to-report process within hours, enabling highly accurate species identification of respiratory pathogens and simultaneous detection of multiple functional genes in a single test.

[0086] In terms of primer design, the primer combinations of the present invention have been subjected to rigorous bioinformatics screening and multiple optimizations in the following aspects to ensure high specificity and high sensitivity of detection: a. Precise target screening and sequence verification: Based on pathogen whole-genome sequence alignment, species-specific conserved regions (such as the viral NS1 gene and the bacterial gyrA gene) are prioritized as targets. BLASTn verification ensures that primers only match the target pathogen, minimizing cross-reactivity risks. Cross-reactivity prediction and homology verification are performed on all primer sequences to avoid primer dimer formation and amplification of non-target sequences, ensuring the specificity and effectiveness of each amplification in the multiplex reaction.

[0087] b. High annealing temperature (Tm) design: To address the complexity of the large number of primers in the multiplex system, a high and uniform annealing temperature (Tm) was strategically selected and uniformly designed. This significantly reduces primer mismatches and nonspecific amplification at non-optimal temperatures, thereby improving the specificity and reliability of the overall amplification reaction.

[0088] c. Strict GC content and secondary structure control: The GC content of all primers is optimized and maintained within a balanced range (40%-60%). Furthermore, professional primer design software (such as Primer-BLAST, OligoAnalyzer, and SnapGene) is used to predict, evaluate, and screen the secondary structures (e.g., hairpin structures and self-dimers) of all primers. This minimizes the risk of primer self-folding or mutual interference, avoids chain termination caused by terminal mismatches, and ensures amplification efficiency.

[0089] Therefore, through the comprehensive design of the above three optimization dimensions (target specificity, Tm uniformity, and structural stability), the primer combination proposed in the embodiment of this application achieves excellent specificity, sensitivity, and amplification efficiency in a complex ultra-multiplex detection system, laying a solid foundation for subsequent high-precision sequencing detection.

[0090] While the present invention is described through the above-described embodiments, the present invention is not limited to the above-described detailed methods, nor does it necessarily rely on the above-described detailed methods for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0091] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0092] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A reagent for identifying respiratory tract infection pathogens and detecting drug resistance and virulence, comprising: A set of multiple primers, each of the multiple primers comprises a first sequence, and the first sequences are shown as SEQ ID NOs: 1-165, respectively. The identification of respiratory tract infection pathogens and the detection of drug resistance and virulence are all carried out in the same primer pool comprising the collection.

2. The reagent according to claim 1, characterized in that The length of the amplicons of the multiple primers ranges from 200 to 2100 bp, and the amplicons are suitable for single-molecule sequencing.

3. The reagent according to claim 1 or 2, characterized in that The content of each primer in the set of multiple primers in the reagent is the same.

4. The reagent according to claim 3, characterized in that Each of the plurality of primers further comprises a second sequence, wherein the second sequence comprises a tag sequence for distinguishing the source of the sample.

5. A kit for identifying respiratory tract infection pathogens and detecting drug resistance and virulence, comprising: The reagent according to any one of claims 1 to 4 and one or more of the following: DNA polymerase, reverse transcriptase, dNTP, reaction buffer, Mg 2+ and single-molecule library construction reagents, including The single-molecule library construction reagents include one or more of the following: a single-molecule sequencing adapter, an end repair reagent, a ligation reagent for the single-molecule sequencing adapter, a purification reagent, and a tag sequence.

6. Use of the reagent according to any one of claims 1 to 4 or the kit according to claim 5 in the preparation of a preparation for identifying pathogens of respiratory tract infections and detecting drug resistance and virulence, the use comprising: a. The reagents are mixed with the nucleic acid sample to be tested to prepare a multiplex PCR reaction solution; b. placing the multiplex PCR reaction solution in a thermal cycler program to obtain an amplicon of the target region of the nucleic acid sample to be tested based on multiple primers; c. single-molecule library construction and sequencing of the amplicon to obtain sequencing data of each amplicon; and d. parsing the sequencing data to identify respiratory tract infection pathogens in the nucleic acid sample and detect drug resistance and virulence, The nucleic acid sample to be tested is DNA or RNA. Based on the fact that the nucleic acid sample to be tested is RNA, before step a, the use further comprises: e. performing reverse transcription on the nucleic acid sample to be tested.

7. The use according to claim 6, characterized in that The thermal cycling program includes: Pre-denaturation stage: 98°C, 2-5 min; Cycling stage: 98°C, 15 s; 62°C, 30 s; 58°C, 30 s, 72°C, 1-5 min, 25-40 cycles; and Supplementary extension phase: 72°C, 5-10 min.

8. The use according to claim 7, characterized in that The working concentration of each of the multiple primers in the multiplex PCR reaction solution is 10 μM, and The amount of nucleic acid sample to be tested is 0.1-100 ng. The detection limit of the respiratory tract infection pathogen in the nucleic acid sample to be tested is 100 copies / μL.

9. The use according to any one of claims 6 to 8, characterized in that The nucleic acid sample to be tested is single or multiple, wherein based on the number of nucleic acid samples to be tested being multiple, step a comprises: The reagents are mixed with each of the nucleic acid samples to be tested to prepare the multiplex PCR reaction solution, wherein the reagents include a set of multiple primers, wherein The primer set comprises a plurality of primers, each of which comprises a first sequence and a second sequence, wherein the first sequences are shown as SEQ ID NOs: 1-165 respectively; and the second sequence comprises a tag sequence for distinguishing the source of a sample.

10. An integrated system for identifying respiratory tract infection pathogens and detecting drug resistance and virulence in multiple nucleic acid samples based on single-molecule sequencing, comprising: i. Amplification - pre-built library module, for: amplifying the target region related to the identification of respiratory tract infection pathogens and the detection of drug resistance and virulence in the nucleic acid sample to be tested by the reagent according to claim 1, to obtain an amplicon of each target region, wherein the reagent comprises a set of multiple primers, wherein The primer set comprises a plurality of primers, each of which comprises a first sequence and a second sequence, wherein the first sequences are respectively as shown in SEQ ID NOs: 1-165, and the second sequence comprises a tag sequence for distinguishing the source of the sample; ii single molecule sequencing adapter introduction module for introducing a single molecule sequencing adapter to one side of each amplicon to obtain a single molecule sequencing library of the nucleic acid sample to be tested; iii. a single molecule sequencing module for performing single molecule sequencing on the single molecule sequencing library to obtain sequencing data of each amplicon; and iv. A data analysis module for analyzing the sequencing data to identify respiratory tract infection pathogens in the nucleic acid sample and detect drug resistance and virulence.

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