Reagent, kit for identifying respiratory pathogen and detecting drug resistance and virulence thereof based on single molecule sequencing method and application thereof

Abstract

The present application relates to the technical field of molecular biology detection, and specifically provides a reagent for identifying respiratory tract infection pathogens and detecting drug resistance and virulence genes, comprising: a plurality of primer sets, each primer in the plurality of primers comprising a first sequence as shown in SEQ ID NO: 1-165 and an optional second sequence for distinguishing sample sources. The target points detected by the super-multiplex primer combination proposed in the present application comprehensively cover the pathogen directory (a total of 353 kinds) of the latest domestic and foreign respiratory tract infection diagnosis guidelines, realize low-cost, high-sensitivity, high-specificity, high-throughput detection, and can effectively and comprehensively guide clinical drug use.

Description

Technical Field

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

[0002] Respiratory infections, a major challenge in global public health, encompass various diseases caused by bacteria, fungi, viruses, and other microorganisms, and occur worldwide, particularly among children, the elderly, and immunocompromised groups. Meanwhile, drug resistance is becoming increasingly prominent, posing a significant challenge in the treatment of respiratory infections. Currently, the diagnosis, treatment, surveillance, and prevention of tuberculosis rely on pathogen identification and various drug susceptibility tests. Commonly used clinical methods include pathogen culture of Mycobacterium tuberculosis, acid-fast staining smear microscopy, molecular biology methods, immunological techniques, and imaging techniques. Among these methods, pathogen culture is the gold standard for tuberculosis diagnosis; however, its long culture period of 2-8 weeks makes it unsuitable for providing rapid diagnostic results and may even delay optimal treatment. Furthermore, general laboratories, due to their lower biosafety levels, cannot perform Mycobacterium tuberculosis culture, thus severely limiting the feasibility and efficiency of pathogen culture. Furthermore, while microscopic detection of Mycobacterium tuberculosis in sputum or other bodily fluids is a faster and simpler method, it suffers from poor sensitivity and cannot predict strain resistance. Immunological techniques, including the interferon-gamma release assay (IGRA), can detect levels of interferon-gamma release specific to Mycobacterium tuberculosis, but these are expensive and cross-react with non-tuberculous mycobacteria, affecting accuracy.

[0003] Molecular biological diagnostic methods, including real-time fluorescence PCR detection technology and sequencing techniques, have emerged. Nucleic acid amplification test (NAAT) is an important reference standard for the diagnosis of respiratory pathogens. Meanwhile, emerging rapid pathogen detection technologies such as metagenomic sequencing (mNGS), single-molecule sequencing (TGS), and targeted sequencing (TS) also provide numerous options for respiratory pathogens. However, while nucleic acid amplification tests (such as GeneXpert® MTB / RIF) can detect Mycobacterium tuberculosis and rifampicin resistance in sputum within 2 hours, like real-time fluorescence PCR, their target number is limited, making it difficult to comprehensively analyze multiple respiratory pathogen species and their resistance. In clinical practice, metagenomic sequencing (including mNGS and mTGS) still faces three major technical bottlenecks when applied to respiratory pathogen detection: First, the proportion of human host nucleic acid in clinical respiratory samples is too high (often exceeding 95%), resulting in dilution of microbial nucleic acid load. Even with targeted capture and other technologies, the sensitivity of pathogen detection remains low. Second, the microbial composition of respiratory specimens is complex, making it difficult to distinguish between colonizing and pathogenic bacteria. For example, the colonization rate of Streptococcus pneumoniae in the nasopharynx of healthy individuals can reach 30%, and its genome coverage and pathogenicity exhibit a non-linear dynamic relationship, making pathogen identification often require a combination of semi-quantitative thresholds and clinical characteristics. More troubling is the lack of universally accepted data interpretation standards for current metagenomic sequencing (including mNGS and mTGS) detection technologies. This leads to unclear relationships between sequencing results and treatment, and the interpretation of massive amounts of data is extremely complex (involving multiple stages such as raw data quality control, sequence alignment, microbial species annotation, gene prediction, and functional annotation), placing high demands on computing resources and analysts.

[0004] Therefore, there is an urgent need to provide a reagent and method for identifying respiratory pathogens and detecting their drug resistance that is rapid, covers a wide range of drug resistance mutations, is simple and easy to use, and is low in cost, with high sensitivity, high accuracy / specificity. Summary of the Invention

[0005] The first aspect of this application provides a reagent for identifying respiratory tract infection pathogens and detecting their drug resistance and virulence, comprising: a set of multiple primers, each of the multiple primers containing a first sequence, the first sequence being as shown in SEQ ID NO: 1-165, wherein the identification of respiratory tract infection pathogens and the detection of their drug resistance and virulence are performed in the same primer pool containing the set.

[0006] In some embodiments, the length of the amplicon from the multiple primers ranges from 200 to 2100 bp, and the amplicon is suitable for single-molecule sequencing.

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

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

[0009] The second aspect of this application also provides a kit for identifying respiratory infection pathogens and detecting their drug resistance and virulence, comprising: reagents as described in any embodiment of the first aspect of this application and one or more of the following: DNA polymerase, reverse transcriptase, dNTPs, reaction buffer, Mg... 2+ And single-molecule library preparation reagents, wherein the single-molecule library preparation reagents include one or more of the following: single-molecule sequencing adapters, end repair reagents, ligation reagents for the single-molecule sequencing adapters, purification reagents, and tag sequences.

[0010] The third aspect of this application also proposes the use of a reagent as described in any embodiment of the first aspect of this application or a kit as described in any embodiment of the second aspect of this application in the preparation of a formulation for the identification of respiratory infectious pathogens and the detection of 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 amplicones 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 amplicones to obtain sequencing data for each amplicon; and d. parsing the sequencing data to identify and detect the drug resistance and virulence of respiratory infectious pathogens in the nucleic acid sample, wherein the nucleic acid sample to be tested is DNA or RNA.

[0011] In some embodiments, where the nucleic acid sample to be tested is RNA, prior to step a, the method further includes: e. performing reverse transcription on the nucleic acid sample to be tested.

[0012] In some embodiments, the thermal cycling program includes: a pre-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, and the amount of the nucleic acid sample to be tested is 0.1-100 ng, and the detection limit of the respiratory 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. When there are multiple nucleic acid samples to be tested, 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 primer including a first sequence and a second sequence, the first sequence being as shown in SEQ ID NO: 1-165; the second sequence including a tag sequence for distinguishing the source of the sample.

[0015] The fourth aspect of this application also proposes an integrated system for identifying respiratory pathogens and detecting drug resistance and virulence in multiple nucleic acid samples based on single-molecule sequencing. The system includes: i. an amplification-pre-library module for amplifying target regions in the nucleic acid samples to be tested related to the identification of respiratory pathogens and the detection of drug resistance and virulence using reagents as described in any embodiment of the first aspect of this application, to obtain amplicons for each target region, wherein the reagents include a set of multiple primers, each primer containing a first sequence and a second sequence, the first sequence being as shown in SEQ ID NO: 1-165, and the second sequence containing a tag sequence for distinguishing the sample origin; ii. a 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 samples 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 respiratory pathogens in the nucleic acid sample and to detect their drug resistance and virulence.

[0016] The technical solution of this application achieves the following technical effects:

[0017] 1. This application's embodiments do not require microbiological culture of the collected clinical respiratory samples. Instead, they directly amplify the nucleic acids of the clinical samples using a multi-target primer combination. The resulting target fragments are compatible with long amplicon sequences from single-molecule long-read sequencers. Compared to traditional detection methods, the primer combination proposed in this application covers a wider range of common respiratory pathogens, their clinically relevant drug resistance and virulence genes, and 16S targets specifically designed for bacterial species. The primer combination covers the pathogen catalogs of the latest domestic and international respiratory infection diagnostic guidelines, as well as emerging pathogens not covered in existing technologies. Utilizing single-molecule nanopore long-read sequencing, this primer combination can cover the V1-V9 regions of 16S rDNA. This full-length sequence detection achieves extremely high annotation at the species level and excellent bacterial classification and species identification capabilities (traditional short-read sequencing only covers the V1-V2, V1-V3, V4, and V3-V5 regions of 16S rDNA). The primer combinations and detection methods proposed in this application can detect nucleic acids at concentrations as low as 1 ng / μL and 100 copies / μL, with a sensitivity of 100% and a specificity of 100%. This significantly improves the sensitivity and accuracy of pathogen detection and provides effective guidance for clinical medication.

[0018] 2. This application's embodiments, through a special design of primer combinations, achieve a streamlined and synergistic primer design. Compared to primer design in traditional next-generation sequencing platforms, this design optimizes primer Tm values, GC content, and secondary structures in multiple dimensions while ensuring primer capture specificity. The designed primer combinations exhibit no cross-reactivity and extremely low mismatch rates in comprehensive detection of common respiratory pathogens, demonstrating high and balanced amplification efficiency and extremely high detection efficiency, especially in terms of sensitivity and specificity. Simultaneously, the special design of multiple primer combinations significantly reduces the demand for templates and detection reagents, and saves approximately 2 hours of tag sequence ligation time in traditional library preparation. Therefore, it reduces detection costs and greatly shortens detection time.

[0019] 3. The multiplex PCR detection method proposed in this application can also achieve simultaneous detection of multiple samples. By combining this detection method with drug resistance result analysis software, an integrated "detection-analysis-reporting" system can be formed. Compared with traditional detection and analysis, this application reduces the entire process turnaround time from nearly 8 hours to about 5 hours, a reduction of approximately 37.5%. Simultaneously, this application can achieve simultaneous detection of multiple samples while reducing experimental time and steps, resulting in faster detection speed, lower chip consumption, higher sensitivity, and stronger specificity. This integrated system enables high-throughput, hourly "sample-to-report" detection with rapid, efficient, and highly accurate results.

[0020] 4. The embodiments of this application can also be equipped with technical analysis software designed specifically for single-molecule sequencing data of each amplicon. For clinical samples, only 600 Mb of sequencing data is generally required to achieve rapid and accurate identification of respiratory pathogens and 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 requirements in traditional analysis and reducing the computing power requirements, making the detection faster, more convenient and economical. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the primer structure according to an embodiment of this application;

[0023] Figure 2 This describes the analysis process for single-molecule sequencing data of samples according to embodiments of this application. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0025] This application creatively designs primer combinations for the identification of respiratory infection pathogens and the one-time detection of multiple drug resistance genes and virulence genes, an integrated library construction scheme for simultaneous detection of multiple samples, and a complete data analysis process for this scheme. It provides a fast, stable, highly sensitive, highly accurate, and low-cost feasible solution that guides the entire process from sample to report.

[0026] The first aspect of this application provides a reagent for identifying respiratory tract infection pathogens and detecting their drug resistance and virulence, comprising: a set of multiple primers, each primer containing a first sequence as shown in SEQ ID NO: 1-165 in Table 1, wherein the identification of respiratory tract infection pathogens and the detection of their drug resistance and virulence are performed in the same primer pool containing the set.

[0027] Table 1

[0028]

[0029] The primer combinations proposed in this application comprehensively cover the pathogen catalogs of the latest domestic and international guidelines for the diagnosis of respiratory infections, encompassing 353 species including bacteria, fungi, DNA viruses, RNA viruses, parasites, mycoplasma, chlamydia, ureaplasma, spirochetes, and rickettsia (see Table 2). This covers epidemiological data on pathogen spectrum reported in core journals in the field of respiratory infections over the past decade. Therefore, using the primer combinations provided in this application enables effective and comprehensive identification of respiratory infection pathogens and guides clinical medication. Furthermore, the special design of the primer combinations in this application achieves a streamlined number of primers with synergistic effects. Compared to primer designs in traditional next-generation sequencing platforms, it optimizes Tm values, GC content, and secondary structures in multiple dimensions while ensuring primer capture specificity. It can achieve synchronous and stable amplification of all targets in ultra-multiplex PCR with 165 primers participating simultaneously, with no cross-reaction and extremely low mismatch rate, exhibiting high and balanced amplification efficiency and extremely high detection efficiency, especially in terms of sensitivity and specificity. The primer combination proposed in this application is designed for single-molecule long-read sequencing platforms, comprehensively covering the V1-V9 regions of the pathogen's 16S rDNA. Compared to traditional short-read sequencing, which only covers the V1-V2, V1-V3, V4, and V3-V5 regions of the 16S rDNA, the primer combination proposed in this application achieves extremely high annotation at the species level and excellent bacterial classification and species identification capabilities. Based on its special design and integration with the single-molecule platform, the primer combination proposed in this application can achieve rapid, accurate, and high-precision identification of respiratory infection pathogens and the one-time detection of comprehensive drug resistance and virulence genes, with detection concentrations as low as 1 ng / μL and 100 copies / μL of sample nucleic acid, with 100% sensitivity and 100% specificity. This significantly improves the sensitivity and accuracy of pathogen detection and can effectively and comprehensively guide clinical medication.

[0030] Table 2: Classification of 353 pathogens

[0031]

[0032] Table 3: Details of 353 Pathogens

[0033]

[0034] In some embodiments, the length of the amplicon from the multiple primers ranges from 200 to 2100 bp, such as 200-400, 300-800, 400-1000, 500-2000 bp, or any value or range between them. These long amplicones are suitable for single-molecule sequencing. In some embodiments, single-molecule sequencing includes nanopore sequencing. In the embodiments of this application, each primer is designed to amplify long amplicon adapted to a single-molecule long-read sequencer, thereby completely covering the V1-V9 region of 16S rDNA, and enabling real-time reading of the entire single-molecule sequence in combination with single-molecule sequencing. This achieves high-throughput, hourly "sample to report" rapid, efficient, and highly accurate pathogen identification and detection of drug resistance and virulence genes.

[0035] In some embodiments, in addition to the first sequence as shown in SEQ ID NO: 1-165, each of the multiple primers also includes a second sequence, which contains a tag sequence (e.g., a barcode) for distinguishing the source of the sample. In some embodiments, the tag sequence may be located on the 5' side of the first sequence (e.g., ...). Figure 1 (As shown). It is understood that the primers used to amplify the same sample carry the same second sequence (tag sequence); the primers used to amplify different samples carry different second sequences (tag sequences). In this embodiment, by introducing tag sequences into each primer, the tag introduction for multi-sample detection in the conventional single-molecule sequencing library construction process can be avoided, saving the operation time of the tag introduction step (about 2 hours), and realizing the one-step "amplification-tag labeling (i.e. library pre-preparation)", thereby greatly simplifying the experimental operation, shortening the detection time, and improving the detection throughput. It is understood that, in addition to the tag sequence, the second sequence may also contain one or more other sequences, such as universal sequences (i.e., fixed sequences) used for enrichment and purification, sequencing adapter recognition / ligation, etc. This application does not limit the type of other sequences contained in the second sequence.

[0036] In some embodiments, the primers in the multiple primer sets have the same content. In some embodiments, the working concentration of each primer is 10 μM or less. The primer combinations proposed in this application, when mixed at equal concentrations, can achieve synchronous and stable amplification of each target site in ultramultiplex PCR with 165 primers participating simultaneously, without cross-reaction and with an extremely low mismatch rate, exhibiting high and balanced amplification efficiency and extremely high detection efficiency, especially in terms of sensitivity and specificity.

[0037] In some embodiments, reagents for identifying respiratory pathogens and detecting drug resistance and virulence genes may also incorporate primers targeting other target genes or target mutation sites, as needed, such as those targeting other respiratory pathogens, like 16S rRNA, IS...6110 Genes such as the 16S-23S rRNA intergenic spacer (ITS) and genes targeting other drug resistance or virulence targets fall within the scope of protection of this application.

[0038] The second aspect of this application provides a kit for identifying respiratory infection pathogens and detecting their drug resistance and virulence, comprising: reagents as described in any embodiment of the first aspect of this application and one or more of the following: DNA polymerase, reverse transcriptase, dNTPs, reaction buffer, Mg... 2+ The kit includes single-molecule library preparation reagents, wherein the single-molecule library preparation reagents comprise one or more of the following: single-molecule sequencing adapters, end-repair reagents, ligation 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 tag sequence, the single-molecule library preparation reagent may also contain a tag 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 an equal concentration. It is understood that, in addition to the above-described 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.

[0039] The third aspect of this application provides a method for identifying respiratory infectious pathogens and detecting their drug resistance and virulence, comprising: a. mixing reagents as described in any embodiment of the first aspect of this application 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 amplicones for 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 amplicones to obtain sequencing data for each amplicon; and d. analyzing the sequencing data to identify respiratory infectious pathogens in the nucleic acid sample and detect their drug resistance and virulence.

[0040] In some embodiments, the nucleic acid sample to be tested is DNA or RNA, wherein, based on the nucleic acid sample to be tested being RNA, the method further includes, prior to step a, e. performing reverse transcription on the nucleic acid sample to be tested.

[0041] 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 an equal concentration.

[0042] In some embodiments, the amount of nucleic acid sample used is 0.1-300 ng, for example, 0.1-100, 0.1-150, 0.1-200 ng or any value or range thereof. In some embodiments, taking a 50 μL system as an example, the multiplex PCR reaction solution may include: a. a reaction mixture, including DNA polymerase, dNTPs, Mg... 2+The primer combinations provided in this application are: a. 25 μL of reaction buffer; b. 10 μL of primer mixture (equal volumes of each primer, 10 μM); c. 1 μL of nucleic acid sample to be tested (concentration 10-250 ng / μL); and d. 50 μL of sterile water. These primer combinations enable highly specific and accurate identification of pathogens and detection of drug resistance and virulence genes with extremely low sample volumes. In some embodiments, the detection limit for respiratory pathogens in the nucleic acid sample to be tested is as low as 100 copies / μL.

[0043] In some embodiments, the thermal cycling program includes: a pre-denaturation stage: 98°C, 2-5 min; a cycling stage: 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 stage: 72°C, 5-10 min; and an optional storage stage: 4°C-10°C.

[0044] In this embodiment, the proportions of each component and primer concentration in the amplification system are adaptively optimized based on primer combinations, reducing cross-reactions between primers and solving compatibility issues in traditional multiplex PCR (such as primer dimers and differences in amplification efficiency). 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 foundation for high-throughput, low-cost, high-sensitivity, and high-accuracy identification of respiratory tract infection pathogens and detection of drug resistance and toxicity.

[0045] In some embodiments, the nucleic acid sample to be tested can be single or multiple. When there are multiple nucleic acid samples to be tested, step a can include: mixing reagents with each nucleic acid sample to be tested to prepare a multiplex PCR reaction solution. The reagents comprise a set of multiple primers, each primer containing a first sequence and a second sequence. The first sequence is shown in SEQ ID NO: 1-165, and the second sequence contains a tag sequence for distinguishing the sample origin. That is, this application proposes a method for identifying respiratory pathogens and detecting drug resistance and virulence in multiple samples. Based on the special design of the tag sequences of each primer, integrated amplification and library construction of multiple samples can be achieved, saving approximately 2 hours of ligation time in traditional library construction. Simultaneously, the requirement for templates and detection reagents for each primer is extremely low, thereby greatly shortening the detection time and reducing detection costs.

[0046] In some embodiments, such as Figure 2As shown, step d: parsing the sequencing data to identify respiratory pathogens in nucleic acid samples and to detect their drug resistance and virulence may include the following steps:

[0047] i. Input long-read targeted amplification data and filter the data using NanoFilt or similar tools;

[0048] ii. Construct your own alignment database based on the target sequences 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 virus database (GenBank, https: / / www.ncbi.nlm.nih.gov / genbank / ), the bacterial resistance database (ResFinder, http: / / genepi.food.dtu.dk / resfinder), and the bacterial virulence database (VFDB, https: / / www.mgc.ac.cn / VFs / ).

[0049] iii. Using the Minimap2 read alignment tool, the filtered high-quality sequences are aligned to the reference sequences or reference genomes of each target to obtain the target coverage and the number of alignments, and the coverage depth is calculated.

[0050] The above process has been developed into the software pathogen_detection.py (software copyright registration number: 2024SR1731913) using a Python 3.10.6 compilation environment, which is available for direct download and use. This tool can comprehensively process targeted sequencing data through an automated workflow, including adapter removal, data quality control, long read sequence alignment, and statistical analysis, thereby providing efficient and accurate identification of respiratory infection pathogens and comprehensive one-time detection of multiple drug resistance and virulence genes. Operation command: python pathogen_detection.py input_path qlt database output_dir (q: specifies the minimum quality threshold for NanoFilt; sequences below this threshold will be filtered out, recommended value 7; l: specifies the minimum length threshold for NanoFilt; sequences shorter than this threshold will be filtered out, recommended value 0; t: specifies the number of Minimap2 alignment threads, recommended values ​​12, 24).

[0051] The method proposed in this application addresses the need for simultaneous multi-sample testing in clinical settings, providing a technical workflow for integrated multi-sample library construction, a database for analyzing the generated data, and a complete analysis scheme. Compared to traditional single-molecule nanopore sequencing library construction methods, integrated multi-sample library construction can shorten the library construction time by approximately 2 hours and enables simultaneous testing of multiple samples while reducing experimental time and steps. It offers faster detection speed, lower chip consumption, higher sensitivity, and stronger specificity. Furthermore, utilizing a self-developed database and systematic tools, this analysis tool can operate efficiently with lower computing resources—for clinical samples, only 600 Mb of sequencing data is required to achieve accurate identification of respiratory pathogens and prediction of drug resistance and virulence genes through this workflow. For a 64-bit CPU, the entire data analysis process can be completed within 10 minutes. Therefore, the method proposed in this application not only significantly shortens the detection time, increases the detection speed, and reduces the data volume requirement, but also reduces the need for high-end computing equipment, making detection faster, more convenient, and more economical.

[0052] The fourth aspect of this application provides for the use of reagents as described in any embodiment of the first aspect of this application or kits as described in any embodiment of the second aspect of this application in the preparation of formulations for the identification of respiratory tract infection pathogens and the detection of drug resistance and virulence.

[0053] The fifth aspect of this application proposes an integrated system for identifying respiratory pathogens and detecting drug resistance and virulence in multiple nucleic acid samples based on single-molecule sequencing, comprising: i. an amplification-pre-library module for amplifying target regions in the nucleic acid samples to be tested related to the identification of respiratory pathogens and the detection of drug resistance and virulence using reagents as described in any embodiment of the first aspect of this application, to obtain amplicons for each target region, wherein the reagents include a set of multiple primers, wherein the set of primers contains multiple primers, each primer containing a first sequence and a second sequence, the first sequence being as shown in SEQ ID NO: 1-165, and the second sequence containing a tag sequence for distinguishing the sample origin; ii. a 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 samples 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. The data analysis module is used to analyze the sequencing data to identify respiratory pathogens in the nucleic acid sample and to detect their drug resistance and virulence.

[0054] In the embodiments of this application, any or all of the steps in the above-mentioned quality control filtering, comparison, 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.

[0055] It should be noted that the explanation and description of the embodiments of "reagent for identification of respiratory tract infection pathogens and detection of drug resistance and virulence" in this application are also applicable to kits containing the reagent, methods for using the reagent, and systems, which have the same technical effects and advantages, and will not be repeated here.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0058] Except for the specific parameters indicated, the data analysis in the following embodiments follows conventional procedures in the art, such as conventional analysis methods and / or default parameters using relevant software.

[0059] Example 1

[0060] This embodiment uses the primer combination shown in SEQ ID NO: 1-165 to test ZYMO D6311 bacterial and fungal DNA standards (ZymoBIOMICS Microbiome Standard (non-uniform species distribution), 220 ng, 20 μL, 11 ng / μL) and their tenfold serially diluted samples (D6311). 1:10 1.1 ng / μL, D6311 1:100 0.11 ng / μL and D6311 1 :1000 The bacteria and fungi in the sample (0.011 ng / μL) were detected to verify the feasibility of the primer combination and detection method proposed in the embodiments of this application. The specific steps are as follows:

[0061] 1.1 Multiplex PCR

[0062] 1.1.1 Mix equal volumes of the primer combinations shown in SEQ ID NO: 1-165 to prepare a 10 μM primer mixture. Prepare multiplex PCR reaction solutions according to the systems shown in Table 4. These solutions are used to amplify ZYMO D6311 bacterial and fungal DNA standards (11 ng / μL) and 1:10 diluted D6311 DNA, respectively. 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):

[0063] Table 4

[0064]

[0065] 1.1.2 Place the prepared multiplex PCR reaction solution in a PCR instrument and amplify using the following thermal cycling program: (1) Pre-denaturation: 98 ℃, 3 min; (2) Denaturation, annealing and extension: 98 ℃, 15 s, 62 ℃, 30 s, 58 ℃, 30 s, 72 ℃, 110 s, 35 cycles; (3) Supplementary extension: 72 ℃, 5 min; (4) Cool down to 4 ℃ and hold.

[0066] 1.1.3 The reaction products were purified using magnetic beads, and the purified reaction products were subjected to quality testing.

[0067] 1.2 Single-sample, single-molecule long-read library construction

[0068] Using the H940-000013 CycloneSEQ Universal Library Prep set (24 RXN), single-molecule library construction was performed on the purified reaction products, strictly following its instruction manual. This included a barcode ligation step that took approximately 2 hours to distinguish different sample sources.

[0069] 1.3 Single-molecule long-read sequencing

[0070] Using the H940-000016 CycloneSEQ WT Sequencing Kit (6T), and strictly following its instruction manual, single-molecule long-read sequencing was performed on the single-molecule library in section 1.2.

[0071] 1.4 Data Analysis

[0072] For reference Figure 2 The analysis process shown uses the software pathogen_detection.py (software copyright registration number: 2024SR1731913) to perform statistical analysis on the offline data.

[0073] 1.5 Results

[0074] 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 amplification product concentration of (0.011 ng / μL) was too low to meet the library construction requirements, therefore no further single-molecule library construction was performed. Meanwhile, as shown in Table 5, the standard stock solution ZYMO D6311 (11 ng / μL) and D6311... 1:10 Effective amplification was achieved at 1.1 ng / μL, and the N50 length (the length of a sequence that, when arranged from longest to shortest and then added together, reaches exactly 50% of the total length) met the design length of the amplicon. This indicates that the primer combination designed in this embodiment can be used to amplify various targets, and the sequencing data volume meets the analysis requirements, demonstrating that the primer combination proposed in this embodiment achieves efficient amplification. Furthermore, this result also suggests that the detection limit of the primer combination in this embodiment is as low as 1.1 ng / μL (which is basically consistent with the requirements of rTaq Multiplex 2X Master Mix for sample nucleic acid).

[0075] The target genes in the ZYMO D6311 bacterial and fungal DNA standards and their tenfold serially diluted samples used in this embodiment mainly include the bacterial 16S rDNA gene. rpoB Genes and fungi ITS region. The species contained 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.

[0076] Table 6 shows the primer pair ZYMO D6311 (11 ng / μL) and D6311 in this embodiment. 1:10 The detection status of each target in (1.1 ng / μL). Table 6 shows the detection status of ZYMO D6311 (11 ng / μL) and its ten-fold serially diluted sample D6311. 1:10 The detection data (1.1 ng / μL) is sufficient to meet the analytical requirements. Based on this data, bacterial and fungal species can be well distinguished, enabling accurate identification of various pathogens. This result also reflects the effectiveness of the multiplex PCR primer combination and the corresponding reaction system and procedure in this embodiment, and demonstrates the technical reliability of the entire process from sample to report.

[0077] Table 5

[0078]

[0079] Table 6

[0080]

[0081] The experimental results above demonstrate that the primer combination in this embodiment exhibits excellent amplification performance when detecting mixed (non-uniformly distributed bacterial and fungal) DNA standards. Each primer can accurately and sensitively (detection limit as low as 1.1 ng / μL) capture specific targets of the target species, laying a solid foundation for subsequent sequencing and analysis. Furthermore, sequencing validation proves that the primer combination and workflow design in this embodiment can be applied to a single-molecule nanopore long-read platform, effectively distinguishing different microbial species and specific targets, providing strong technical support for in-depth research on microbial communities.

[0082] Example 2

[0083] This embodiment uses primer combinations containing sequences as shown in SEQ ID NO: 1-165 (i.e., the first sequence) and sample tag sequences (i.e., the second sequence, randomly selected from link barcodes LB01-LB24 in H940-000018 Cyclone SEQ 24 Barcode Library Prep Set (4 RXN), with #2 tag LB02 and #6 tag LB06 used in this embodiment) to detect bacterial and fungal targets in ZYMO D6311 bacterial and fungal DNA standards (ZymoBIOMICS Microbiome Standard (non-uniform species distribution), 220 ng, 20 μL, 11 ng / μL), and establishes a multi-sample library construction and sequencing workflow. The specific steps are as follows:

[0084] 2.1 Multiplex PCR

[0085] Referring to the method in step 1.1 of Example 1, multiple standard samples were amplified using primer combinations containing the first and second sequences described above, wherein the second sequences of the primer combinations used for different samples were different, namely, #2 tag and #6 tag.

[0086] 2.2 Multi-sample single-molecule long-read library construction

[0087] Using the H940-000013 CycloneSEQ Universal Library Prep set (24 RXN), single-molecule library construction was performed on the purified reaction products strictly following its documentation, omitting the sample tag (barcode) linking step.

[0088] 2.3 Single-molecule long-read sequencing

[0089] Refer to step 1.3 of Example 1.

[0090] 2.4 Data Analysis

[0091] Refer to step 1.4 of Example 1.

[0092] 2.5 Results

[0093] Sample tags #2 and #6 were randomly selected and used to label the sequencing samples of two sets of ZYMO D6311 amplicones, respectively. Table 7 shows the concentration of amplified products and single-molecule long-read sequencing data statistics of the primer combinations with sample tag sequences in this application. As can be seen from Table 7, the primer combinations with sample tag sequences also showed stability in amplifying the target region, and all achieved effective amplification of the target region. This indicates that the primer combinations designed in this embodiment can be used to simultaneously amplify each target site of multiple samples, while the sequencing data volume meets the analysis requirements, demonstrating that the primer combinations proposed in this embodiment achieve efficient amplification.

[0094] Table 8 shows the detection results of each target in ZYMO D6311 using the primer combination of this embodiment. As can be seen from Table 8, guided by the primer combination with the sample tag sequence, the detection data for ZYMO D6311 is sufficient to meet the analytical requirements. Based on this data, bacterial and fungal species can be well distinguished, achieving accurate identification of various pathogens. This result also reflects the effectiveness of the ultra-multiplex PCR primer combination and its corresponding reaction system and procedure in this embodiment, and demonstrates the technical reliability of the integrated multi-sample detection process from sample to report.

[0095] Table 7

[0096]

[0097] Table 8

[0098]

[0099] The experimental results above demonstrate that the primer combination proposed in this embodiment exhibits excellent amplification performance when detecting mixed (non-uniformly distributed bacteria and fungi) DNA standards of multiple species. Each primer can accurately and sensitively capture the specific targets of the target species, laying a solid foundation for subsequent sequencing and analysis. Furthermore, sequencing validation proves that the primer combination and workflow design of this embodiment can be applied to the single-molecule nanopore long-read platform, effectively distinguishing different microbial species and specific targets, providing strong technical support for in-depth research on microbial communities. Simultaneously, using the primer combination of this embodiment for amplification eliminates the two-hour tag ligation step in traditional library construction, thereby achieving a high level of adaptation between the multi-sample integrated library construction system and the single-molecule nanopore long-read sequencing platform, with faster detection speed, lower chip consumption, higher sensitivity, and stronger specificity.

[0100] Example 3

[0101] This embodiment uses primer combinations containing sequences as shown in SEQ ID NO: 1-165 (i.e., the first sequence) and sample tag sequences (i.e., the second sequence, randomly selected from link barcodes LB01-LB24 in the H940-000075 Cyclone SEQ 24 Barcode Library Prep Set (4 RXN), with #1 tag LB01 and #2 tag LB02 used in this embodiment) to test the capture and analysis of target regions in mixed samples containing bacteria and fungi under the multi-sample integrated analysis workflow established in Example 2, in order to evaluate the detection capability of the primer combinations proposed in this embodiment for multi-species samples. The mixed samples containing bacteria and fungi consist of ATCC MSA-4000 bacterial DNA standard (non-uniform species distribution, approximately 1 ng / μL) and ATCC MSA-1010 fungal DNA standard, which correspond to 12 target sequences and 10 fungal targets, respectively. Both samples comprehensively encompass bacterial species, fungal species, and bacterial resistance genes. The specific steps are as described in Example 2. The sequencing results of two groups, sequencing tags #1 and #2, were randomly selected and are shown in Tables 9 and 10.

[0102] Table 9 shows the concentrations of amplified products and the statistics of single-molecule long-read sequencing data for samples #1 and #2 after sequencing. As can be seen from Table 9, the amplified product concentrations of each sample guided by the primer combination proposed in this embodiment are high. Under the integrated library construction process incorporating the tag sequence, the constructed library quality is high, and sequencing can generate sufficient data. This amount of data can fully meet the analysis requirements, indicating that the primer combination proposed in this embodiment achieves efficient amplification based on the reaction system and reaction procedure of this embodiment, and performs excellently in the integrated library construction process based on it.

[0103] Table 10 shows the detection results of the primer combination in this embodiment for 22 target regions in a mixed sample containing bacteria and fungi. As can be seen from Table 10, guided by the primer combination with sample tag sequences, the amplification, library construction, and sequencing process of this embodiment can effectively capture and analyze each target sequence in the sample, achieving a 100% detection rate. Based on these data, it is possible to distinguish between different bacterial and fungal species, achieving accurate identification of various pathogens. Furthermore, the sequencing results of the "multi-sample integrated library construction" method can also effectively distinguish between different samples, reflecting the effectiveness of the ultra-multiplex PCR reaction system and the "multi-sample integrated library construction" method in this embodiment, and demonstrating the technical reliability of the multi-sample integrated detection process from sample to report.

[0104] Table 9

[0105]

[0106] Table 10

[0107]

[0108] The experimental results above demonstrate that the primer combination proposed in this embodiment exhibits excellent amplification performance when detecting mixed (non-uniformly distributed bacteria and fungi) DNA standards of multiple species. Each primer can accurately and sensitively capture the specific targets of the target species, laying a solid foundation for subsequent sequencing and analysis. Furthermore, sequencing validation proves that the primer combination and workflow design of this embodiment can be applied to the single-molecule nanopore long-read platform, effectively distinguishing different microbial species and specific targets, providing strong technical support for in-depth research on microbial communities. Simultaneously, using the primer combination of this embodiment for amplification eliminates the two-hour tag ligation step in traditional library construction, thereby achieving a high level of adaptation between the multi-sample integrated library construction system and the single-molecule nanopore long-read sequencing platform, with faster detection speed, lower chip consumption, higher sensitivity, and stronger specificity.

[0109] Example 4

[0110] This embodiment uses primer combinations containing sequences as 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:

[0111] 4.1 Multiplex PCR

[0112] Referring to the method in step 1.1 of Example 1, primer combinations containing sequences as shown in SEQ ID NO: 1-165 (i.e., the first sequence) and sample tag sequences (i.e., the second sequence) were used to amplify the following samples: sputum, bronchoalveolar lavage fluid, pleural effusion, and pharyngeal swabs. All four types of samples were provided by Zhongshan Hospital affiliated with Fudan University, and the pathogen detection results from the clinical microbiology laboratory showed non-tuberculous mycobacteria as the predominant symptom. In the reaction system, the amount of bronchoalveolar lavage fluid sample added was 1 μL, and its initial sample concentration is shown in Table 11.

[0113] 4.2-4.4 Multi-sample single-molecule long-read library construction

[0114] 4.5 Results

[0115] Table 11 shows the original nucleic acid concentration, amplification product concentration, and subsequent sequencing data volume and analysis results for each sample in this embodiment. As can be seen from Table 11, the primer combination proposed in this embodiment can efficiently amplify each target sequence of the original sample, and the amount of data produced is sufficient to meet the needs of subsequent analysis. Furthermore, the positive species identified based on the primer combination and multi-sample analysis process of this embodiment are consistent with the prior results, indicating that the primer combination and process of this embodiment are accurate in identifying each species in the sample. In addition, as can be seen from Table 11, this embodiment can effectively detect each pathogen even with a sample concentration as low as 0.344 ng / μL (see number 15), requiring an average data volume of only about 500 Mb, demonstrating the high sensitivity, high accuracy, and extremely low computational requirements of the single-molecule sequencing-based primer combination and process proposed in this embodiment in clinical sample analysis.

[0116] Table 11

[0117]

[0118] The experimental results above demonstrate that the primer combination proposed in this application exhibits excellent performance in identifying respiratory pathogens and amplifying drug resistance and virulence targets in clinical samples. The primers can accurately, sensitively, and specifically capture various designed respiratory pathogen targets, thereby achieving targeted amplification of the targets to be detected. This meets the needs for detecting various pathogens in clinical samples, including viruses, bacteria, fungi, parasites, and atypical pathogens, as well as detecting key functional genes such as drug resistance genes. Sequencing results also indicate that the primer combination and detection method designed in this application can be used for identifying various respiratory pathogens in clinical respiratory samples and detecting their drug resistance and virulence genes. Furthermore, by using specially designed primers, the approximately two-hour barcode introduction step in the multi-sample library preparation process can be omitted, thus significantly shortening the detection time. Furthermore, the primer combinations and detection methods proposed in this application can be adapted to multi-sample integrated library construction systems and single-molecule nanopore long-read sequencing platforms. They have faster detection speeds, lower chip consumption, higher sensitivity, stronger specificity, and achieve high-accuracy detection (consistent with clinical pathogen detection results).

[0119] Industrial applicability

[0120] This application creatively designs a super-multiplex primer combination (containing 165 primer sequences) for the identification of 353 respiratory tract infection pathogens and the detection of drug resistance and virulence genes. Based on this, it employs a multi-sample, high-efficiency library construction method and sequencing data analysis workflow. Through super-multiplex PCR amplification of target genes under appropriate reaction systems and procedures, construction of single-molecule long sequencing libraries from the amplicon, and single-molecule sequencing and data analysis of these libraries, accurate, sensitive, and comprehensive identification of respiratory tract infection pathogens and the detection of their drug resistance and virulence genes are achieved. Through the special design of the primer combination and detection workflow, the primer combination and detection method proposed in this application achieve low-cost, high-sensitivity, high-specificity, and high-throughput detection. Simultaneously, it features a short detection cycle, high accuracy, and comprehensive gene detection, fully covering the pathogen spectrum epidemiological data reported in core journals in the field of respiratory infections over the past decade. This enables effective and comprehensive identification of respiratory tract infection pathogens and guides clinical medication.

[0121] In summary, compared to conventional pathogen detection methods, the specially designed primer combinations proposed in this application have the following core advantages:

[0122] 1. Broader target coverage and higher clinical value: The primer combinations proposed in this application break through the limitations of general pathogen detection, integrating five major categories of respiratory infection-related pathogens (including viruses, bacteria, fungi, parasites, and atypical pathogens) and key functional genes (such as drug resistance genes), thereby enhancing pathogen identification and infection mechanism analysis capabilities from multiple dimensions. This not only reduces the false negative rate but also simultaneously guides clinical precision medicine decisions.

[0123] 2. More streamlined design and more stable performance: Compared with primer design schemes based on next-generation sequencing (NGS), this scheme uses fewer primers, which effectively reduces the risk of primer dimer formation and non-specific amplification, improves reaction efficiency, reduces reagent consumption, and lowers the cost per test.

[0124] 3. Highly compatible with long-read sequencing platforms for rapid and accurate detection: 165 specially designed primers with optimized amplicon lengths are perfectly adapted to single-molecule long-read sequencing platforms. Based on this approach, a rapid workflow from sample to report can be achieved in "hours," enabling high-precision identification of respiratory pathogen species and simultaneous detection of multiple functional genes in a single test.

[0125] In terms of primer design, the primer combinations in this application embodiment have undergone rigorous bioinformatics screening and multiple optimizations to ensure high specificity and high sensitivity of the detection:

[0126] a. Precise Target Screening and Sequence Validation: Based on pathogen whole-genome sequence alignment, species-specific conserved regions (such as the viral NS1 gene and the bacterial gyrA gene) are preferentially selected as targets. BLASTn validation is used to ensure that primers only match the target pathogen, avoiding the risk of cross-reaction. Cross-reaction 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 multiplex reactions.

[0127] b. High annealing temperature (Tm) design: To address the complexity of the large number of primers in multiplex systems, a high and uniform annealing temperature (Tm) was strategically selected and designed. This significantly reduced primer mismatches and nonspecific amplification at non-optimal temperatures, thereby improving the specificity and reliability of the overall amplification reaction.

[0128] c. Strict GC content and secondary structure control: The GC content of all primers was optimized and maintained within a balanced range (40%-60%). Simultaneously, professional primer design software (such as Primer-BLAST, OligoAnalyzer, SnapGene, etc.) was used to predict, evaluate, and screen the secondary structures (such as hairpin structures and self-dimers) of all primers. This minimized the risk of primer self-folding or mutual interference, avoided chain termination effects caused by end mismatches, and ensured amplification efficiency.

[0129] Therefore, through the comprehensive design of the above three optimization dimensions (target specificity, Tm uniformity, and structural stability), the primer combination proposed in this application embodiment achieves excellent specificity, sensitivity, and amplification efficiency in a complex ultramultiplex detection system, laying a solid foundation for subsequent high-precision sequencing detection.

[0130] The present invention has been illustrated with the above embodiments to explain the detailed method of the present invention. However, the present invention is not limited to the detailed method described above, that is, it does not mean that the present invention must rely on the detailed method described above to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0131] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0132] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A reagent for identifying respiratory infection pathogens and detecting their drug resistance and virulence, comprising: A collection of multiple primers, each primer containing a first sequence, as shown in SEQ ID NO: 1-165 respectively. The identification of respiratory infection pathogens and the detection of drug resistance and virulence are all performed in the same primer pool containing the set.

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

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

4. The reagent according to claim 3, characterized in that, Each of the multiple primers also contains a second sequence, which contains a tag sequence for distinguishing the source of the sample.

5. A kit for identifying respiratory infection pathogens and detecting their drug resistance and virulence, comprising: The reagent as described in any one of claims 1 to 4 and one or more of the following: DNA polymerase, reverse transcriptase, dNTPs, reaction buffer, Mg 2+ and single-molecule library preparation reagents, among which The single-molecule library preparation reagent includes one or more of the following: single-molecule sequencing adapter, end repair reagent, ligation reagent of the single-molecule sequencing adapter, purification reagent, and tag sequence.

6. Use of the reagent of any one of claims 1 to 4 or the kit of claim 5 in the preparation of formulations for the identification of respiratory tract infection pathogens and the detection of drug resistance and virulence, said use comprising: a. Mix the reagents with the nucleic acid sample to be tested to prepare a multiplex PCR reaction solution; b. Place the multiplex PCR reaction solution in a thermal cycling program to obtain amplicon of the target region of the nucleic acid sample to be tested based on multiple primers; c. Perform single-molecule library construction and sequencing on the amplicon to obtain sequencing data for each amplicon; and d. Analyze the sequencing data to identify respiratory pathogens in the nucleic acid samples and to detect their drug resistance and virulence. The nucleic acid sample to be tested is either DNA or RNA. If the nucleic acid sample to be tested is RNA, prior to step a, the purpose further includes: e. Perform reverse transcription on the nucleic acid sample to be tested.

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

8. The use according to claim 7, characterized in that, The working concentration of each primer in the multiplex PCR reaction solution is 10 μM, and The amount of nucleic acid sample used for testing is 0.1-100 ng. The detection limit for the respiratory 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 can be single or multiple. Where, based on multiple nucleic acid samples to be tested, step a includes: The reagents are mixed with each of the nucleic acid samples to be tested to prepare the multiplex PCR reaction solution, wherein the reagents comprise a set of multiple primers. The primer set comprises multiple primers, each primer containing a first sequence and a second sequence, the first sequence being as shown in SEQ ID NO: 1-165, and the second sequence containing a tag sequence for distinguishing the source of the sample.

10. An integrated system for identifying respiratory pathogens and detecting drug resistance and virulence in multiple nucleic acid samples based on single-molecule sequencing, the system comprising: i. An amplification-pre-library module, used to: amplify target regions in the nucleic acid sample to be tested related to the identification of respiratory pathogens and the detection of drug resistance and virulence using the reagents as described in claim 1, to obtain amplicones for each target region, wherein the reagents comprise a set of multiple primers, wherein The primer set includes multiple primers, each primer containing a first sequence and a second sequence, the first sequence being as shown in SEQ ID NO: 1-165, and the second sequence containing a tag sequence for distinguishing the source of the sample; ii. A single-molecule sequencing adapter introduction module, used to introduce a single-molecule sequencing adapter to one side of each of the amplicon to obtain a single-molecule sequencing library of the nucleic acid sample to be tested; iii. A single-molecule sequencing module, used to perform single-molecule sequencing on the single-molecule sequencing library to obtain sequencing data of each amplicon; and iv. A data parsing module for parsing the sequencing data to identify respiratory pathogens in the nucleic acid samples and to detect their drug resistance and virulence.

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