A method for targeted sequencing of multiple pathogenic microorganisms
By combining a triple-targeted enrichment system and an anti-inhibition multiplex PCR system with a bioinformatics analysis module, we have achieved precise typing of pathogenic microorganisms, detection of drug resistance genes, and analysis of virulence genes. This solves the problems of accuracy and speed in pathogenic microorganism detection in existing technologies and improves detection efficiency and result accuracy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JINHUA VOCATIONAL TECH COLLEGE
- Filing Date
- 2026-03-12
- Publication Date
- 2026-06-19
AI Technical Summary
Existing technologies cannot accurately type pathogenic microorganisms, detect drug resistance genes, or analyze virulence genes. The analysis process relies on general databases, which is not specific and is slow.
By employing a triple-targeted enrichment system and an anti-inhibition multiplex PCR system, combined with a bioinformatics analysis module, pathogen typing, drug resistance gene detection, and virulence gene analysis can be completed simultaneously.
It significantly improves detection efficiency, meets the clinical needs for rapid, accurate, and comprehensive pathogen detection, simplifies the operation process, improves the accuracy and stability of test results, and reduces the difficulty of operation.
Smart Images
Figure CN122235283A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a targeted sequencing method for various pathogenic microorganisms. Background Technology
[0002] Pathogenic microbial infection refers to the process by which microorganisms such as bacteria, viruses, fungi, and parasites invade and multiply in the human body, causing disease. This has a significant impact on human health and daily life. Given the increasingly serious threat of pathogenic microorganisms, accurate and rapid detection of these microorganisms is of great importance.
[0003] Chinese patent CN120249523A discloses a targeted sequencing method for multiple pathogenic microorganisms, specifically including the following steps: collecting biodetection materials containing pathogenic microorganisms; extracting nucleic acids from the pathogenic microorganisms to be detected; designing specific primers and probes based on the target pathogenic microorganisms; amplifying the nucleic acid sequences using PCR technology; performing deep sequencing using a high-throughput sequencer; and performing bioinformatics analysis on the detection results. By designing specific primers and probes, the method can efficiently enrich the nucleic acid sequences of the target pathogenic microorganisms. The use of high-throughput sequencing technology for deep sequencing significantly improves the sensitivity and accuracy of detecting multiple pathogenic microorganisms. It can simultaneously detect multiple different types of pathogenic microorganisms such as bacteria, viruses, and fungi. This method is simple to operate, time-saving, and low-cost, enabling rapid and accurate identification of pathogenic microorganisms and providing strong support for timely and effective prevention and control measures.
[0004] While the aforementioned patents can achieve simultaneous detection of multiple pathogenic microorganisms in practical use, they can only identify pathogen species and cannot perform precise pathogen typing, drug resistance gene detection, or virulence gene analysis. Furthermore, the analysis process relies on general databases, resulting in poor targeting and slow analysis speed. Therefore, they do not meet current needs. To address this, we propose a targeted sequencing method for multiple pathogenic microorganisms. Summary of the Invention
[0005] The purpose of this invention is to provide a targeted sequencing method for multiple pathogenic microorganisms. Through optimized sample pretreatment, high-quality nucleic acid templates are provided for subsequent targeted enrichment and PCR amplification. The triple targeted enrichment system provides high-purity target nucleic acids for PCR amplification. The anti-inhibition multiplex PCR system ensures efficient amplification of target nucleic acids in complex samples. High-throughput sequencing enables rapid sequencing of target nucleic acids. The bioinformatics analysis module enables simultaneous completion of pathogen typing, drug resistance gene detection, and virulence gene analysis, which greatly improves the overall detection efficiency and can meet the clinical needs for rapid, accurate, and comprehensive pathogenic microorganism detection, thus solving the problems mentioned in the background art.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a targeted sequencing method for multiple pathogenic microorganisms, comprising the following steps:
[0007] S1: Rapid nucleic acid extraction and impurity removal are performed on clinical samples in an integrated process to obtain purified pathogenic microbial nucleic acid;
[0008] S2: A triple-targeted enrichment system is used to target and enrich purified nucleic acids to obtain enriched pathogenic microorganism target nucleic acids;
[0009] S3: Using an anti-inhibition multiplex PCR system, the target nucleic acid is amplified by multiplex PCR to obtain the PCR amplification product;
[0010] S4: Library construction and high-throughput sequencing of PCR amplification products to obtain raw sequencing data;
[0011] S5: Employs a bioinformatics analysis module to analyze raw sequencing data, simultaneously completing pathogen typing, drug resistance gene detection, and virulence gene analysis, and outputting a test report.
[0012] Preferably, the implementation process of S1 specifically includes:
[0013] Clinical samples were taken, added to a compound enzymatic digestion system, and incubated at 37-42℃ for 15-25 minutes to achieve pathogen cell lysis, host protein degradation, and removal of contaminating RNA. The compound enzymatic digestion system consists of lysozyme, proteinase K, and RNase inhibitors.
[0014] Add specific magnetic beads and binding buffer to the incubated system, and incubate with shaking at room temperature for 5-10 minutes to allow the nucleic acids of bacteria, viruses, and fungi to bind to the specific magnetic beads simultaneously.
[0015] Place the mixture on a magnetic rack to attract magnetic beads, and discard the supernatant;
[0016] Add washing buffer to the magnetic beads on the magnetic rack, shake and wash 2-3 times. After each wash, the magnetic beads will be adsorbed and the supernatant will be discarded.
[0017] Add elution buffer to the washed magnetic beads, incubate at 55-65℃ for 5-10 minutes, shake to mix, place on a magnetic rack to adsorb the magnetic beads, and collect the supernatant, which is the purified pathogenic microbial nucleic acid.
[0018] Preferably, the clinical sample is selected from one or more of the following: blood, cerebrospinal fluid, sputum, urine, feces, pharyngeal swabs, and bronchoalveolar lavage fluid.
[0019] Preferably, the triple-targeted enrichment system in S2 includes specific primers, specific probes, and specific nucleic acid aptamers; wherein, the specific primers are used to specifically amplify the target nucleic acids of pathogenic microorganisms, the specific probes are used to specifically identify, enhance, and block the signal of the target nucleic acids of pathogenic microorganisms, and the specific nucleic acid aptamers are used to accurately identify specific antigens or conserved regions of nucleic acids on the surface of pathogenic microorganisms.
[0020] Preferably, the anti-inhibition multiplex PCR system in S3 includes PCR buffer, anti-inhibition reagent, specific primers, Taq enzyme, dNTPs, and target nucleic acid; wherein the anti-inhibition reagent is a compound reagent of BSA and betaine.
[0021] Preferably, the high-throughput sequencing implementation process in S4 specifically includes:
[0022] A sequencing library preparation kit was used to prepare the PCR amplification products into a library, including end repair, A-tailing, adapter ligation, and PCR enrichment, to obtain the sequencing library.
[0023] The sequencing libraries were quality checked by using agarose gel electrophoresis to detect library fragment size and Qubit to detect library concentration.
[0024] The qualified sequencing library was loaded onto the high-throughput sequencing platform for sequencing.
[0025] Preferably, the implementation process of S5 specifically includes:
[0026] The raw sequencing data is filtered to remove low-quality reads, adapter sequences, and host genome sequences, resulting in clean reads.
[0027] Clean reads are compared with a dedicated pathogen database, and cluster analysis is performed using deep learning algorithms to achieve accurate pathogen typing.
[0028] Clean reads are compared with drug resistance gene databases to identify drug resistance gene sequences, analyze the drug resistance spectrum of pathogens, and clarify drug resistance mechanisms;
[0029] Clean reads are compared with a virulence gene database to screen out virulence gene sequences, which can help clinically determine the severity of infection.
[0030] The integrated analysis results output a test report that includes pathogen species, typing, drug resistance status, virulence gene information, and test reliability.
[0031] Preferably, the precise typing includes at least one of serological typing and genotyping, with typing resolution reaching the strain level, capable of distinguishing different subtypes of pathogenic microorganisms of the same species.
[0032] Preferably, the bioinformatics analysis module includes a dedicated pathogen database, a drug resistance gene database, a virulence gene database, and a deep learning analysis algorithm.
[0033] Preferably, the analysis process of the bioinformatics analysis module is optimized for triple-target enriched sequencing data, which can effectively filter host nucleic acid sequences and non-target nucleic acid sequences, reduce redundant data interference, and improve analysis speed.
[0034] Compared with the prior art, the beneficial effects of the present invention are:
[0035] 1. The triple-targeted enrichment system of this invention adds a specific nucleic acid aptamer. The three components work synergistically against the same pathogenic microorganism target, with non-overlapping and complementary target sites, forming a novel targeted enrichment mechanism. Specifically, the nucleic acid aptamer can accurately identify specific antigens or conserved nucleic acid regions on the surface of pathogenic microorganisms, specifically binding to low-abundance pathogen nuclei and avoiding interference from host nucleic acids. This significantly improves the capture capacity of low-abundance pathogens, greatly increases the utilization rate of sequencing data, and reduces the cost of invalid sequencing. This invention optimizes the design of specific primers, probes, and nucleic acid aptamers to maximize their synergistic effect, further improving the specificity and efficiency of targeted enrichment and avoiding false positives caused by non-specific binding.
[0036] 2. This invention constructs an anti-inhibition multiplex PCR system. By adding a BSA and betaine complex anti-inhibition reagent, it can specifically bind to inhibitors in clinical samples, eliminating their impact on PCR amplification. Through terminal base modification and cross-species conserved region screening, primer dimer formation is reduced, while improving primer compatibility with pathogenic microbial variants. It can cover the main variant subtypes of pathogenic microorganisms, greatly improving the clinical adaptability of the method. The use of hot-start Taq enzyme further inhibits non-specific amplification, improves the stability and accuracy of PCR amplification, ensures the reliability of detection results for complex clinical samples, and can meet the detection needs of different types of clinical samples.
[0037] 3. This invention achieves simultaneous completion of pathogen typing, drug resistance gene detection, and virulence gene analysis through a bioinformatics analysis module, filling the gap of the single function of existing bioinformatics analysis technology, greatly improving detection efficiency, avoiding the overuse of antibiotics and antiviral drugs, and virulence gene analysis can predict the risk of infection in advance, helping to assist in the early intervention of severe infections, and significantly improving the clinical practical value of the method.
[0038] 4. This invention optimizes the sample pretreatment process, achieving rapid extraction of pathogenic nucleic acids and removal of impurities in one step. The complex enzymatic digestion system consists of lysozyme, proteinase K, and RNase inhibitors, which can rapidly lyse pathogenic cells, degrade host proteins and contaminating RNA. Specific magnetic beads can simultaneously bind to the nucleic acids of bacteria, viruses, and fungi, eliminating the need for step-by-step extraction, simplifying the operation process, effectively reducing the interference of residual impurities on subsequent PCR amplification and sequencing processes, and further improving the accuracy and stability of the detection results. At the same time, the integrated pretreatment process facilitates standardized operation in clinical laboratories, reduces operational difficulty, and is conducive to the clinical promotion and application of the method. Attached Figure Description
[0039] Figure 1 This invention provides a targeted sequencing method for multiple pathogenic microorganisms;
[0040] Figure 2 This invention provides a targeted sequencing method for multiple pathogenic microorganisms. Detailed Implementation
[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0042] To address the limitations of existing technologies, which only enable pathogen species-level identification and cannot perform precise pathogen typing, drug resistance gene detection, or virulence gene analysis, and whose analysis processes rely on general databases, resulting in poor specificity and slow analysis speed, please refer to [link to relevant documentation]. Figures 1-2 This embodiment provides the following technical solution:
[0043] A targeted sequencing method for multiple pathogenic microorganisms includes the following steps:
[0044] S1: Rapid nucleic acid extraction and impurity removal are performed on clinical samples in an integrated process to obtain purified pathogenic microbial nucleic acid;
[0045] S2: A triple-targeted enrichment system is used to target and enrich purified nucleic acids to obtain enriched pathogenic microorganism target nucleic acids;
[0046] S3: Using an anti-inhibition multiplex PCR system, the target nucleic acid is amplified by multiplex PCR to obtain the PCR amplification product;
[0047] S4: Library construction and high-throughput sequencing of PCR amplification products to obtain raw sequencing data;
[0048] S5: Employs a bioinformatics analysis module to analyze raw sequencing data, simultaneously completing pathogen typing, drug resistance gene detection, and virulence gene analysis, and outputting a test report.
[0049] The implementation process of S1 specifically includes:
[0050] Clinical samples were taken and added to a compound enzymatic digestion system. The samples were incubated at 37-42℃ for 15-25 minutes to achieve pathogen cell lysis, host protein degradation, and removal of contaminating RNA. The compound enzymatic digestion system consisted of lysozyme, proteinase K, and RNase inhibitor. The final concentration of lysozyme was 10-20 mg / mL, the final concentration of proteinase K was 5-10 mg / mL, and the final concentration of RNase inhibitor was 0.5-1 U / μL.
[0051] Add specific magnetic beads and binding buffer to the incubated system, and incubate with shaking at room temperature for 5-10 minutes to allow the nucleic acids of bacteria, viruses, and fungi to bind to the specific magnetic beads simultaneously. The specific magnetic beads are magnetic beads with nucleic acid binding groups modified on their surface. The binding buffer is a Tris-HCl buffer with a pH of 7.0-7.5, containing 50-100 mmol / L NaCl and 0.5-1% Tween-20.
[0052] Place the mixture on a magnetic rack to attract magnetic beads, and discard the supernatant;
[0053] Add washing buffer to the magnetic beads on the magnetic rack, shake and wash 2-3 times. After each wash, adsorb the magnetic beads and discard the supernatant. The washing buffer is a Tris-HCl buffer with pH 7.0-7.5 containing 100-150 mmol / L NaCl, 0.1-0.5% Tween-20 and 5-10% ethanol.
[0054] Add elution buffer to the washed magnetic beads, incubate at 55-65℃ for 5-10 minutes, shake to mix, place on a magnetic rack to adsorb the magnetic beads, and collect the supernatant, which is the purified pathogenic microbial nucleic acid; the elution buffer is a Tris-HCl buffer with pH 8.0-8.5 containing 1-5 mmol / L EDTA.
[0055] Clinical samples are selected from one or more of the following: blood, cerebrospinal fluid, sputum, urine, feces, throat swabs, and bronchoalveolar lavage fluid.
[0056] The S2 triple-target enrichment system includes specific primers, specific probes, and specific nucleic acid aptamers. These three components work synergistically against the same pathogenic microorganism target, with mutually exclusive and complementary target sites. Specifically, the specific primers are used to specifically amplify the target nucleic acid of the pathogenic microorganism, the specific probes are used to specifically identify the target nucleic acid of the pathogenic microorganism, enhance the signal, and block non-target nucleic acid. The specific nucleic acid aptamers are used to accurately identify specific antigens or conserved regions of nucleic acids on the surface of pathogenic microorganisms and specifically bind to low-abundance pathogenic nucleic acids to avoid interference from host nucleic acids.
[0057] The S3 anti-inhibition multiplex PCR system includes PCR buffer, anti-inhibition reagent, specific primers, Taq enzyme, dNTPs, and target nucleic acid. The anti-inhibition reagent is a compound reagent of BSA and betaine, with a final concentration of BSA of 0.1-0.5 mg / mL and a final concentration of betaine of 0.5-1.5 mol / L. It can specifically bind to inhibitors in clinical samples and eliminate their influence on PCR amplification.
[0058] The implementation process of high-throughput sequencing in S4 specifically includes:
[0059] A sequencing library preparation kit was used to prepare the PCR amplification products into a library, including end repair, A-tailing, adapter ligation, and PCR enrichment, to obtain the sequencing library.
[0060] The sequencing libraries were quality checked by agarose gel electrophoresis to detect the size of the library fragments (fragment size 200-500bp) and by Qubit to detect the concentration of the library (concentration ≥10ng / μL).
[0061] The qualified sequencing library was loaded onto a high-throughput sequencing platform for sequencing. The sequencing platform was selected from Illumina NovaSeq, Illumina MiSeq or Nanopore MinION. The sequencing mode was paired-end sequencing or single-end sequencing, and the sequencing depth was ≥100×.
[0062] The implementation process of S5 specifically includes:
[0063] The raw sequencing data is filtered to remove low-quality reads (≥50% of bases with a Q value <20), adapter sequences, and host genome sequences, resulting in clean reads.
[0064] Clean reads are compared with a dedicated pathogen database, and clustering analysis is performed using deep learning algorithms to achieve accurate pathogen typing. The dedicated pathogen database covers more than 1,000 common clinical pathogens and variants, including bacteria, viruses, fungi, mycoplasma, chlamydia, etc., and the database is updated regularly.
[0065] Clean reads are compared with drug resistance gene databases to identify drug resistance gene sequences, analyze the drug resistance spectrum of pathogens, and clarify drug resistance mechanisms; the drug resistance gene database contains 500+ drug resistance sites, covering various types such as antibiotic resistance and antiviral drug resistance;
[0066] Clean reads are compared with a virulence gene database to screen out virulence gene sequences, which can help clinically determine the severity of infection.
[0067] The integrated analysis results output a test report that includes pathogen species, typing, drug resistance status, virulence gene information, and test reliability.
[0068] Precise typing includes at least one of serological typing and genotyping, with typing resolution reaching the strain level, which can distinguish between different subtypes of pathogenic microorganisms of the same species.
[0069] S5 can simultaneously detect drug resistance genes of pathogenic microorganisms. The drug resistance genes include at least one of antibiotic resistance genes, antiviral drug resistance genes, and antifungal drug resistance genes, and can identify the type, mutation site, and corresponding drug resistance mechanism of the drug resistance gene.
[0070] Step S5, the bioinformatics analysis module, can screen virulence genes of pathogenic microorganisms. These virulence genes include those related to pathogen invasiveness, toxin production, and host immune escape. Based on the type and expression level of virulence genes, clinical judgment of infection severity and prognosis can be aided.
[0071] Bioinformatics analysis takes no more than 30 minutes, which is more than 80% faster than existing general database analysis processes.
[0072] The S5 pathogen typing accuracy rate is no less than 98%, which can accurately distinguish between pathogens of different serotypes and genotypes of the same species, providing a basis for the precise selection of clinical treatment plans.
[0073] The S5 has an accuracy rate of no less than 95% in detecting drug resistance genes and can detect more than 500 drug resistance sites, covering drug resistance genes corresponding to commonly used clinical antibiotics, antiviral drugs, and antifungal drugs.
[0074] The bioinformatics analysis module can automatically integrate the analysis results of pathogen typing, drug resistance genes, and virulence genes, generate standardized test reports, and clearly indicate the credibility of each analysis indicator, making it easy for clinicians to interpret quickly.
[0075] The bioinformatics analysis module includes a dedicated pathogen database, drug resistance gene database, virulence gene database, and deep learning analysis algorithms, which can simultaneously complete pathogen typing, drug resistance gene detection, and virulence gene analysis.
[0076] The bioinformatics analysis module's analysis workflow is optimized for triple-target enriched sequencing data, effectively filtering host nucleic acid sequences and non-target nucleic acid sequences, reducing redundant data interference, and improving analysis speed.
[0077] The present invention provides a targeted sequencing method for multiple pathogenic microorganisms, which adds a specific nucleic acid aptamer when constructing a triple-targeted enrichment system. The three aptamers work synergistically against the same pathogenic microorganism target, and the target sites do not overlap and are complementary, thus forming a novel targeted enrichment mechanism. Among them, nucleic acid aptamers can accurately identify specific antigens or conserved regions of nucleic acids on the surface of pathogenic microorganisms, specifically binding to low-abundance pathogen nuclei and avoiding interference from host nucleic acids, thereby significantly improving the capture capacity of low-abundance pathogens, greatly increasing the utilization rate of sequencing data, and reducing the cost of invalid sequencing. Optimization of the design of specific primers, probes, and nucleic acid aptamers ensures maximum synergistic effects, further improving the specificity and efficiency of targeted enrichment and avoiding false positives caused by non-specific binding. When constructing an anti-inhibition multiplex PCR system, a BSA and betaine complex anti-inhibition reagent is added, which can specifically bind to inhibitors in clinical samples, eliminating their impact on PCR amplification. Terminal base modification and cross-species conserved region screening reduce primer dimer formation and improve primer compatibility with pathogenic microorganism variants, covering the main variant subtypes of pathogenic microorganisms and significantly improving the clinical suitability of the method. The use of hot-start Taq enzyme further inhibits non-specific amplification, improving the stability and accuracy of PCR amplification and ensuring the detection of complex clinical samples. The reliability of the test results can meet the testing needs of different types of clinical samples. Through the bioinformatics analysis module, pathogen typing, drug resistance gene detection, and virulence gene analysis can be completed simultaneously, filling the gap of the single function of existing bioinformatics analysis technology, greatly improving the detection efficiency, avoiding the overuse of antibiotics and antiviral drugs, and virulence gene analysis can predict the risk of infection in advance, helping the early intervention of severe infections, significantly improving the clinical practical value of the method. The sample pretreatment process is optimized, realizing the integration of rapid extraction of pathogen nucleic acid and impurity removal. The compound enzymatic digestion system is composed of lysozyme, proteinase K and RNase inhibitor, which can rapidly lyse pathogen cells, degrade host proteins and contaminating RNA. Specific magnetic beads can bind to the nucleic acids of bacteria, viruses and fungi at the same time, eliminating the need for step-by-step extraction, simplifying the operation process, effectively reducing the interference of residual impurities on the subsequent PCR amplification and sequencing process, and further improving the accuracy and stability of the test results. At the same time, the integrated pretreatment process facilitates standardized operation in clinical laboratories, reduces the difficulty of operation, and is conducive to the clinical promotion and application of the method.
[0078] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0079] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention.
Claims
1. A targeted sequencing method for multiple pathogenic microorganisms, characterized in that, Includes the following steps: S1: Rapid nucleic acid extraction and impurity removal are performed on clinical samples in an integrated process to obtain purified pathogenic microbial nucleic acid; S2: A triple-targeted enrichment system is used to target and enrich purified nucleic acids to obtain enriched pathogenic microorganism target nucleic acids; S3: Using an anti-inhibition multiplex PCR system, the target nucleic acid is amplified by multiplex PCR to obtain the PCR amplification product; S4: Library construction and high-throughput sequencing of PCR amplification products to obtain raw sequencing data; S5: Employs a bioinformatics analysis module to analyze raw sequencing data, simultaneously completing pathogen typing, drug resistance gene detection, and virulence gene analysis, and outputting a test report.
2. The targeted sequencing method for multiple pathogenic microorganisms according to claim 1, characterized in that, The implementation process of S1 specifically includes: Clinical samples were taken, added to a compound enzymatic digestion system, and incubated at 37-42℃ for 15-25 minutes to achieve pathogen cell lysis, host protein degradation, and removal of contaminating RNA. The compound enzymatic digestion system consists of lysozyme, proteinase K, and RNase inhibitors. Add specific magnetic beads and binding buffer to the incubated system, and incubate with shaking at room temperature for 5-10 minutes to allow the nucleic acids of bacteria, viruses, and fungi to bind to the specific magnetic beads simultaneously. Place the mixture on a magnetic rack to attract magnetic beads, and discard the supernatant; Add washing buffer to the magnetic beads on the magnetic rack, shake and wash 2-3 times. After each wash, the magnetic beads will be adsorbed and the supernatant will be discarded. Add elution buffer to the washed magnetic beads, incubate at 55-65℃ for 5-10 minutes, shake to mix, place on a magnetic rack to adsorb the magnetic beads, and collect the supernatant, which is the purified pathogenic microbial nucleic acid.
3. The targeted sequencing method for multiple pathogenic microorganisms according to claim 2, characterized in that, The clinical samples were selected from one or more of the following: blood, cerebrospinal fluid, sputum, urine, feces, throat swabs, and bronchoalveolar lavage fluid.
4. The targeted sequencing method for multiple pathogenic microorganisms according to claim 1, characterized in that, The triple-targeted enrichment system in S2 includes specific primers, specific probes, and specific nucleic acid aptamers. The specific primers are used to specifically amplify the target nucleic acids of pathogenic microorganisms, the specific probes are used to specifically identify, enhance, and block the signal of the target nucleic acids of pathogenic microorganisms, and the specific nucleic acid aptamers are used to accurately identify specific antigens or conserved regions of nucleic acids on the surface of pathogenic microorganisms.
5. The targeted sequencing method for multiple pathogenic microorganisms according to claim 1, characterized in that, The S3 anti-inhibition multiplex PCR system includes PCR buffer, anti-inhibition reagent, specific primers, Taq enzyme, dNTPs, and target nucleic acid; wherein the anti-inhibition reagent is a compound reagent of BSA and betaine.
6. The targeted sequencing method for multiple pathogenic microorganisms according to claim 1, characterized in that, The high-throughput sequencing implementation process in S4 specifically includes: A sequencing library preparation kit was used to prepare the PCR amplification products into a library, including end repair, A-tailing, adapter ligation, and PCR enrichment, to obtain the sequencing library. The sequencing libraries were quality checked by using agarose gel electrophoresis to detect library fragment size and Qubit to detect library concentration. The qualified sequencing library was loaded onto the high-throughput sequencing platform for sequencing.
7. The targeted sequencing method for multiple pathogenic microorganisms according to claim 3, characterized in that, The implementation process of S5 specifically includes: The raw sequencing data is filtered to remove low-quality reads, adapter sequences, and host genome sequences, resulting in clean reads. Clean reads are compared with a dedicated pathogen database, and cluster analysis is performed using deep learning algorithms to achieve accurate pathogen typing. Clean reads are compared with drug resistance gene databases to identify drug resistance gene sequences, analyze the drug resistance spectrum of pathogens, and clarify drug resistance mechanisms; Clean reads are compared with a virulence gene database to screen out virulence gene sequences, which can help clinically determine the severity of infection. The integrated analysis results output a test report that includes pathogen species, typing, drug resistance status, virulence gene information, and test reliability.
8. The targeted sequencing method for multiple pathogenic microorganisms according to claim 7, characterized in that, The precise typing includes at least one of serological typing and genotyping, with typing resolution reaching the strain level, capable of distinguishing different subtypes of pathogenic microorganisms of the same species.
9. The targeted sequencing method for multiple pathogenic microorganisms according to claim 1, characterized in that, The bioinformatics analysis module includes a dedicated pathogen database, a drug resistance gene database, a virulence gene database, and a deep learning analysis algorithm.
10. The targeted sequencing method for multiple pathogenic microorganisms according to claim 9, characterized in that, The bioinformatics analysis module optimizes the analysis process for triple-target enriched sequencing data, effectively filtering host nucleic acid sequences and non-target nucleic acid sequences, reducing redundant data interference, and improving analysis speed.