Liquid hybridization probe pool, kit, and combined detection method for multiple pathogens and drug resistance genes

Abstract

This invention discloses a liquid-phase hybridization probe pool for the joint detection of multiple pathogens and drug resistance genes, designed according to the target regions of the target pathogens and drug resistance genes. The probes are of varying lengths from 30 to 100 nt, including target-specific sequences complementary to the target region and conjugate sequences complementary to adjacent probes. The 5' and 3' ends of the probes are modified with conjugate sequences A and B, respectively, enabling specific binding between adjacent probes through end-pairing. Conjugate sequence A is shown in SEQ ID No. 601, and conjugate sequence B is shown in SEQ ID No. 602. This invention utilizes library construction and specific probe pool hybridization to shorten the capture time to 30 min, significantly outperforming traditional methods. This optimization not only reduces operation time and complexity and minimizes human error but also maintains high sensitivity and specificity, enabling efficient capture of target nucleic acids in complex samples, providing high-quality templates for subsequent sequencing, and supporting the joint detection of multiple respiratory pathogens and drug resistance genes.

Description

Technical Field

[0001] This invention relates to a probe pool, kit, and combined detection method for multiple pathogens and drug resistance genes based on improved liquid-phase hybridization capture technology, belonging to the field of medical molecular diagnostic technology. Background Technology

[0002] Respiratory infections pose a significant global public health challenge, particularly threatening the elderly, infants, and immunocompromised populations. Approximately 70%–80% of upper respiratory tract infections are caused by viruses, including coronaviruses, influenza viruses, parainfluenza viruses, and respiratory syncytial viruses; lower respiratory tract infections are caused by a variety of microorganisms, including viruses, bacteria, and chlamydia. Currently, pathogen identification primarily relies on routine microbiological tests (CMTs), such as culture, smears, PCR, and serological testing. However, these methods are cumbersome, time-consuming, and have limited detection rates, and are not very effective in detecting rare or difficult-to-culture pathogens.

[0003] While multiplex PCR and digital PCR can achieve multi-target detection, their primer design is inflexible, requiring workflow optimization to adjust targets, and they struggle to address pathogen mutations and tracing needs. RNA viruses, in particular, are highly mutable, and probes targeting single subtypes are prone to missing variants. For example, the sequence differences between different adenovirus subtypes are significant, making broad coverage difficult with traditional methods.

[0004] Currently, bacterial resistance testing primarily relies on assay (AST), but this method is culture-dependent and time-consuming, failing to meet the rapid diagnostic needs of acute and severe infections. Existing disease control testing systems (such as culture, mNGS, MLST, and multiplex PCR) also suffer from insufficient detection methods, difficulty in mutation tracking, and limitations in identifying difficult-to-culture bacteria. While metagenomic sequencing (mNGS) enhances the detection capabilities of pathogens and drug resistance genes, it is limited by high costs and a high host background (≥90%), making it unsuitable for low-load samples and fine typing.

[0005] Therefore, there is an urgent need to develop a method and kit for the joint detection of multiple pathogens and drug resistance genes to achieve efficient joint detection and support viral mutation monitoring and tracing. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for joint detection of multiple pathogens and drug resistance genes based on liquid phase hybridization capture technology, which aims to solve the problems of low detection efficiency, limited coverage and inability to monitor viral mutations in real time in the existing technology.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

[0008] A liquid-phase hybridization probe pool for the joint detection of multiple pathogens and drug resistance genes is disclosed. The probes in the liquid-phase hybridization probe pool are designed according to the target regions of the target pathogens and drug resistance genes. The probes are of unequal length, ranging from 30 to 100 nt. Each probe includes a target-specific sequence complementary to the target region of the target pathogen and a conjugate sequence that binds to adjacent probes. The principle is that the 5' and 3' ends of the probes are modified with unique conjugate sequences A and B, respectively. Specifically, the 5'-terminal conjugate sequence A of one probe is inversely complementary to the 3'-terminal conjugate sequence B of another probe, thereby enabling specific binding between adjacent probes through end-to-end pairing. The conjugate sequences are as follows: the 5'-terminal conjugate sequence A is shown in SEQ ID No. 601, and the 3'-terminal conjugate sequence B is shown in SEQ ID No. 602.

[0009] Preferred among these, the target pathogen is one or more of the following: novel coronavirus, influenza A virus, adenovirus, Mycobacterium tuberculosis, Aspergillus fumigatus, Enterococcus faecalis, Streptococcus pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Listeria monocytogenes, Enterobacter cloacae, Pseudomonas aeruginosa, Neisseria meningitidis, Klebsiella pneumoniae, Acinetobacter baumannii, Chlamydia pneumoniae, respiratory syncytial virus, human parainfluenza virus, and influenza B virus.

[0010] The drug resistance genes are Erm (37), rpoB, and rpoB2 genes of Mycobacterium tuberculosis, kpnF and OXA-83 genes of Acinetobacter baumannii, and kpnF, KPC-2, acrR, and ompk37 genes of Klebsiella pneumoniae.

[0011] Among them, the target regions of novel coronavirus are the whole genome; the target regions of influenza A virus are the full length of the HA gene and the full length of the NA gene; the target region of adenovirus is the hexon gene; the target region of drug resistance gene is 150-200 bp in length; and the target region of common respiratory pathogens is 500 bp in length.

[0012] The target-specific sequences of the probes in the liquid-phase hybridization probe pool are shown in SEQ ID No. 1 to SEQ ID No. 600.

[0013] A method for designing the above-mentioned liquid-phase hybridization probe cell includes the following steps:

[0014] (1) Input the initial sequence information of the pathogen and design parameters: The sequence information includes the total sequence information and the target sequence information; the design parameters are: set the hybridization temperature range, probe sequence length range and conjugate probe length range according to the GC content of the target sequence, and the probe adopts an unequal length design;

[0015] (2) Subsequence truncation and statistics: Extract all subsequences of length k from the positive strand and complementary strand sequences of the sum sequence, and count the number of occurrences of each subsequence to provide data support for the selection of conjugate probes;

[0016] (3) Conjugate sequence selection: Based on the design parameters set in step (1), select conjugate sequences of length k;

[0017] (4) Target-specific sequence selection: Select each target sequence sequentially, starting from i=1; select target-specific sequences starting from the nth base of each target sequence to ensure that the binding of the probe to the nucleic acid target sequence is highly specific;

[0018] (5) Probe sequence construction: The conjugate sequence obtained in step (3) is added to the 5' end and 3' end of the target-specific sequence to construct the probe; where i, n and k are positive integers.

[0019] Preferably, the total sequence information is obtained by extracting all possible sequences from the pre-capture library; the target sequence information is obtained by clearly identifying the sequence to be captured and avoiding low-specificity sequences such as repetitive sequences.

[0020] Preferably, the conjugate sequence satisfies the following condition:

[0021] (1) The hybridization temperature of the conjugated sequence is lower than the hybridization temperature at which the probe binds to the target sequence;

[0022] (2) The number of times the conjugate sequence appears in the total sequence is less than 10% of the average.

[0023] A method for joint detection of multiple pathogens and drug resistance genes based on liquid-phase hybridization capture technology includes the following steps:

[0024] (1) Construct a probe pool according to the above method. By controlling and adjusting the number of bases of different pathogen probes, probes of unequal length are generated so that the hybridization temperature of all capture probes is as consistent as possible. The purpose is to improve the hybridization capture efficiency and shorten the reaction time while ensuring capture specificity.

[0025] (2) The probe also includes base complementary conjugated probes. Through the complementary binding of conjugated probes between adjacent reactions, the probe binding is more stable, which further improves the reaction specificity.

[0026] (3) Extract nucleic acid from the sample to be tested and construct a DNA and RNA co-library;

[0027] (4) Using a liquid-phase hybridization capture system, the library obtained in step (2) is hybridized and captured with the probe cell in step (1);

[0028] (5) Amplify, purify and perform high-throughput sequencing on the captured products;

[0029] (6) Bioinformatics analysis: Based on sequencing data, respiratory pathogens, drug resistance genes and novel coronavirus genome variations are identified simultaneously.

[0030] The above probe pool is used in the preparation of novel coronavirus mutation tracking and tracing kits, Mycobacterium tuberculosis, Acinetobacter baumannii and Klebsiella pneumoniae drug resistance gene detection kits and / or other pathogen detection kits mentioned above.

[0031] A kit for implementing a method for joint detection of multiple pathogens and drug resistance genes based on liquid-phase hybridization capture technology, the kit comprising: the probe pool described above; liquid-phase hybridization capture reagent; DNA / RNA co-construction library reagent; and nucleic acid extraction reagent.

[0032] Compared with the prior art, the present invention has the following technical effects:

[0033] (1) This invention achieves efficient detection of multiple subtype pathogens by designing probes targeting key regions for identification and typing, and can track the variation patterns of pathogens, thereby enabling pathogen tracing. This invention significantly improves the detection sensitivity and specificity of multiple pathogens by designing probes of unequal length targeting key typing regions of pathogens and combining them with conjugated sequence structures. Figures 2A to 2D The experimental data shown indicate that, compared to traditional isochronous probe systems, this invention significantly improves the detection sensitivity of pathogens within a 30-minute hybridization time. As shown in Table 4, the introduction of the conjugated sequence further enhances the stability of probe binding. In the detection of COVID-19, the addition of the conjugated sequence significantly increases the number of effective reads and greatly improves the detection sensitivity. For RNA viruses with high variability, by controlling the length of the probe's target region, the probe is placed in a conserved target region, allowing the probe to radiate into regions with high variability through the conserved region, thereby improving both probe utilization and probe capture efficiency.

[0034] (2) This invention employs a specific probe covering the entire genome (approximately 29.9 kb) of the novel coronavirus, enabling efficient whole-genome capture and mutation tracking. For example... Figures 7A to 7D As shown, the target rate of the four clinical samples was ≥90%, the coverage of 0.2×mean was ≥98%, the coverage of 0.5×mean was ≥89%, the average sequencing depth after deduplication was 142, the data were homogeneous, and whole genome assembly and variant analysis were successfully achieved, supporting precise tracing and epidemic prevention and control.

[0035] (3) This invention employs a DNA / RNA co-extraction and co-library construction strategy to optimize the sample processing flow, making it suitable for various clinical sample types (pharyngeal swabs, bronchoalveolar lavage fluid, sputum, etc.). As shown in Table 31, the Q30 of different sample types is ≥90%, the effective reading rate is ≥99%, and the human background interference is low (≤50%), indicating that the method provided by this invention still has excellent performance in samples with high host background. (4) This invention has the ability to detect multiple pathogens and drug resistance genes in an integrated manner. As shown in Tables 28-30, bacteria, viruses, fungi, and drug resistance genes can be detected simultaneously in mixed infection samples, with a positive concordance rate generally reaching 100%. In Example 7, the drug resistance gene detection results were completely consistent with the phenotypic drug sensitivity results, verifying its reliability in drug resistance monitoring.

[0036] (5) The probe pool design avoids cross-hybridization with non-target sequences, resulting in a clean background. For example... Figure 4 As shown, no microorganisms were detected in the negative control (NC) sample, and the Q30 reached 81.07%, indicating a high percentage of effective readings and strong system specificity with no significant non-specific capture.

[0037] (6) For the processing of respiratory samples, this invention employs a DNA and RNA co-extraction kit, which can efficiently extract DNA and RNA simultaneously from respiratory samples, ensuring that all types of nucleic acid components in the sample are completely preserved. Subsequently, a library construction kit is used to construct libraries from the extracted nucleic acids. This process not only optimizes the operation procedure but also significantly improves the efficiency and quality of library construction, laying a solid foundation for subsequent pathogen detection and analysis. Through this integrated sample processing and library construction method, libraries containing information on multiple pathogens and drug resistance genes can be constructed quickly and accurately, thereby achieving co-detection and precise analysis of multiple respiratory pathogens and drug resistance genes.

[0038] (7) In this invention, by further optimizing the liquid-phase hybridization capture process, efficient co-detection of multiple pathogens and drug resistance genes is achieved. By using the constructed library and specific probe pool for hybridization, the capture time is shortened to 30 min, which is significantly better than traditional methods. This optimization not only reduces operation time and complexity and reduces human error, but also maintains high sensitivity and specificity, enabling efficient capture of target nucleic acids in complex samples, providing high-quality templates for subsequent sequencing, and supporting the joint detection of multiple respiratory pathogens (including bacteria, fungi, and viruses) and drug resistance genes. Attached Figure Description

[0039] Figure 1 This is a schematic diagram illustrating the probe design principle in an embodiment of the present invention;

[0040] Figure 2A , Figure 2B , Figure 2C , Figure 2D The graph shows a comparison of the detection sensitivity of different pathogens (Enterococcus faecalis, Serratia marcescens, Escherichia coli, Staphylococcus aureus) using the probe system provided by this invention and a traditional probe system, demonstrating that the present invention has higher detection sensitivity within a 30-minute hybridization time.

[0041] Figure 3 This is a flowchart of a method for joint detection of drug resistance genes of multiple pathogens based on liquid phase hybridization capture technology;

[0042] Figure 4 The sequencing data quality assessment graph shows the Q30 value and percentage of valid reads for each sample, demonstrating that the present invention has high data quality and low background interference.

[0043] Figure 5 The positive concordance rate of different samples at various concentration gradients is statistically shown, demonstrating that the present invention has high sensitivity and consistency in the detection of multiple pathogens.

[0044] Figure 6 The positive concordance rate of each pathogen at different concentration gradients is presented as a statistical chart to further verify the detection capability of this invention for different pathogens.

[0045] Figure 7A The graph shows the target hit rate of the novel coronavirus samples, indicating that the target hit rate is ≥90%.

[0046] Figure 7B Statistical plot of sequencing coverage uniformity for novel coronavirus samples (0.2× Mean and 0.5× Mean coverage);

[0047] Figure 7C The image shows the mean sequencing depth of novel coronavirus samples after deduplication, indicating good data uniformity.

[0048] Figure 7D This is a schematic diagram of the complete genome coverage and assembly results of the novel coronavirus, demonstrating that the present invention can achieve whole genome detection and mutation tracking. Detailed Implementation

[0049] The technical content of the present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0050] Example 1: Design of a liquid-phase hybridization probe cell for joint detection of multiple pathogens and drug resistance genes provided by the present invention.

[0051] This invention designs specific targets targeting conserved regions of common respiratory pathogens. These targets can accurately identify a variety of respiratory pathogens, ensuring high specificity and sensitivity in detection. This makes them suitable for rapid and accurate pathogen detection and identification, providing strong support for the diagnosis and treatment of respiratory infections.

[0052] The challenge of detecting multiple pathogens using liquid-phase hybridization capture technology lies in the diverse range of respiratory pathogens, their varying target regions, and the high variability of many pathogens, especially RNA viruses. This makes traditional detection methods, such as designing probes for the variable regions of a single viral subtype, prone to false negatives. For example, probes targeting a single subtype may fail to effectively detect variant subtypes, affecting the accuracy and timeliness of diagnosis. Furthermore, adenovirus detection faces similar issues; different adenovirus subtypes exhibit significant differences in their gene sequences, making it difficult for traditional detection methods to simultaneously cover multiple subtypes. This invention aims to provide an improved probe design method that designs probes targeting key regions for identification and typing, achieving efficient detection of multiple pathogen subtypes and enabling the tracking of pathogen mutation patterns. Simultaneously, the large number of probes in a multi-pathogen detection probe pool and inconsistent hybridization temperatures result in low hybridization capture efficiency and significant capture difficulty. All probes in this invention are designed with unequal lengths based on the differences in pathogen target region sequences, ranging from 30 to 100 nt in length, and incorporate conjugated sequence fragments, improving reaction efficiency and shortening the hybridization capture time to 30 minutes.

[0053] Specifically, the present invention proposes a liquid-phase hybridization capture probe pool for the joint detection of multiple pathogens and drug resistance genes. The liquid-phase hybridization probe pool contains capture probes covering the pathogens. The probes are designed according to the target region of the target pathogen and are arranged in a non-overlapping manner. Figure 1 This is a schematic diagram illustrating the probe design principle in an embodiment of the present invention. Figure 1 The probe design principles include: designing probes for pathogens with different degrees of variation. Region A has high pathogen polymorphism and requires a high number of probes; Region B has moderate pathogen polymorphism, and designing probes for this region has high versatility. By radiating polymorphic sequences from conserved regions, rich pathogen information can be obtained, and the probes have high versatility.

[0054] The probe is a complex conjugated probe, comprising a target-specific sequence complementary to the target pathogen and drug resistance gene target region, and a conjugated sequence that binds to adjacent probes through complementary pairing. The principle lies in the unique conjugated sequences A and B modified at the 5' and 3' ends of the probe, respectively. Specifically, the 5' end conjugated sequence A of one probe is anticomplementary to the 3' end conjugated sequence B of another probe, enabling specific binding between adjacent probes through end-pairing. The conjugated sequences are as follows: 5' end conjugated sequence A is GTCAGTAGCCG, and 3' end conjugated sequence B is CAGTCATGGC. The probe is also attached to a biotinylate marker, which binds to streptavidin on magnetic beads. The probe fragment length is between 30 and 100 nt.

[0055] The pathogen names, target genes, and NCBI gene IDs in this invention are shown in Table 1.

[0056] Table 1

[0057]

[0058] The probe pool design method provided by this invention, targeting the aforementioned pathogens and drug resistance genes, is as follows:

[0059] 1. Input the initial sequence information and design parameters into the design tool to generate probe sequence information.

[0060] (1) Initial sequence information includes total sequence information, which is extracted from the reference genome database to include all possible sequences;

[0061] (2) Target sequence information: Identify the sequence to be captured. Select regions with a variation of less than 5% for the target sequence and avoid low-specificity sequences such as repetitive sequences, for example, sequences containing more than 10 repetitive bases;

[0062] (3) Design parameters: The hybridization temperature range, probe sequence length range and conjugate sequence length range are set according to the GC content of the target sequence, and the probe adopts an unequal length design.

[0063] The challenge of this invention lies in the design of probes with unequal lengths. Because this invention detects multiple pathogens and drug resistance genes, and the same virus involves different subtype detections, simultaneously detecting so many pathogens and drug resistance genes presents significant challenges. If probes of the same length were of different target sequences, their varying GC contents would lead to substantial differences in hybridization temperatures. This invention employs an unequal-length probe design. For targets with high GC content, short probes can be designed to avoid excessively high Tm values; for targets with low GC content, long probes can be designed to compensate for excessively low Tm values. By adjusting the probe lengths, the final Tm values ​​of all probes tend to be consistent. Thus, under the same reaction conditions, all probes can operate with similar efficiency, resulting in higher hybridization capture efficiency and more accurate results.

[0064] In this embodiment of the invention, a formula that takes GC concentration into account is used to calculate the Tm value of the DNA sequence. The formula is as follows:

[0065] Tm=81.5+0.41\times(\%G+\%C)-\frac{675}{N}+16.6\times\log_{10}(0.05)

[0066] Where N is the length of the target sequence: for example, GTAGTCCGGGCGTCTTCTTTGACTCCGACAAAGGT. According to this formula, the Tm value is 66.12℃. By controlling the GC content, the Tm value can be stabilized.

[0067] Taking several common respiratory viruses, bacteria and fungi as examples, the hybridization temperature was determined based on the calculated TM value. By comparing the hybridization temperatures, it was shown that at 60℃ after 30 min of hybridization, the highest number of sequences were obtained from sequencing the same pathogen, indicating good capture efficiency. The results are shown in Table 2.

[0068] Table 2

[0069]

[0070] 2. Subsequence Extraction and Statistics:

[0071] Extract all subsequences of length k from the positive and complementary chain sequences of the sum sequence, and count the occurrence of each subsequence to provide data support for the selection of subsequent conjugate sequences;

[0072] 3. Selection of conjugate sequences:

[0073] Based on the design parameters set in step 1, select conjugate sequences of length k that satisfy the following conditions:

[0074] (1) The hybridization temperature of the conjugated sequence is lower than the hybridization temperature at which the probe binds to the target sequence;

[0075] (2) The number of times the conjugate sequence k appears in the total sequence is less than 10% of the average.

[0076] The conjugated sequence is a base-complementary conjugated fragment. Through the complementary binding of conjugated probes between adjacent reactions, the probe binding is more stable, further improving the reaction specificity.

[0077] Based on the above design method, this invention designed four sets of sequences as reference conjugate sequences, as follows. Using the IDT OligoAnalyzer tool for comparative analysis, the first set shows Tm values ​​of ~30℃ for both the 5' and 3' tails, which is considered moderately low temperature, conducive to stable hybridization at room temperature or slightly higher temperatures, while avoiding non-specific binding. The second set has higher Tm values ​​(~40℃), which may cause the probe to remain bound at lower temperatures, increasing the risk of non-specific binding. A Tm value that is too low (~20℃) may lead to unstable hybridization, affecting detection sensitivity. The fourth set has inconsistent Tm values ​​(two values ​​for the 5' tail, ~50℃ for the 3' tail), which may lead to asymmetric hybridization, affecting probe performance. The first set has a GC content of 50%, a typical "golden ratio," ensuring hybridization stability while avoiding non-specific binding or secondary structures that may be caused by high GC. The second set has a GC content as high as 70%, easily forming stable non-specific binding or self-dimers. The third set has a GC content of only 20%, which may lead to unstable hybridization. The fourth set has inconsistent GC contents (50% and 60%), which may affect consistency. The low specificity risk in the first group indicates a lower risk of non-specific binding or cross-reactivity. The 5' and 3' end sequence lengths, Tm, and GC content of the first group are symmetrical and consistent, which is beneficial to probe stability and reproducibility. This invention preferentially uses the parameters of the first group for probe design.

[0078] Table 3

[0079]

[0080] The conjugated sequences provided by this invention include a 5' end conjugated sequence A, where the 5' end of one probe is complementary to that of another probe, and a 3' end conjugated sequence B, where the 3' end of one probe is complementary to that of another probe. The 5' end conjugated sequence A used in this invention is GTCAGTACCG, and the 3' end conjugated sequence B is CAGTCATGGC. Intermolecular forces are formed between adjacent probes through base complementarity, improving the binding efficiency between probes and enhancing the system's detection sensitivity.

[0081] This invention also compared the sensitivity of using non-conjugated probes with that of conjugated probes, showing that conjugated probes can improve detection sensitivity. Specifically, using the SARS-CoV-2 genome as a reference genome, two sets of probes were designed. One set of probes was designed according to the description of this system, including both target-specific and conjugated sequences. The other set of probes only included the target-specific sequence. These two sets of probes were used to detect SARS-CoV-2, and the effect of adding or not adding conjugated sequences on virus detection sensitivity was compared. Libraries were constructed using a DNA and RNA co-construction kit (NadPrep® RNA & DNA co-construction module, catalog number: 1002411) from NadPrep (Nanjing) Biotechnology Co., Ltd. Hybridization capture was then performed using probe pools designed in both ways, using the μCaler® Hybrid Capture Reagents v2 hybridization capture kit (catalog number: 1005102). Sequencing was performed, and the number of pathogen sequences detected in different samples was analyzed. The results showed that adding conjugated sequences to the designed probes resulted in a higher number of pathogen sequences detected. Adding conjugated sequences can improve pathogen detection sensitivity, as shown in Table 4.

[0082] Table 4

[0083]

[0084] 4. Target-specific sequence selection:

[0085] Select each target sequence sequentially (starting from i=1). Starting from the nth base of each target sequence (starting from n=1), select target-specific sequences to ensure that the binding of the probe to the nucleic acid target sequence is highly specific.

[0086] 5. Probe sequence construction:

[0087] The conjugate sequences obtained in step 3 are added to the 5' and 3' ends of the target-specific sequence to construct the liquid-phase hybridization probe pool.

[0088] In this embodiment of the invention, the length of the pathogen target area covered by the probe pool is specifically shown in Table 5.

[0089] Table 5

[0090]

[0091] The target region length of common pathogenic microorganisms in the table above is 500 bp. Research data shows that the longer the target region sequence, the higher the probe capture efficiency.

[0092] Using Staphylococcus aureus, Acinetobacter baumannii, and Enterococcus faecalis as examples, probes were designed targeting 100 bp and 500 bp, respectively. Three clinical samples were serially diluted 10-fold, 100-fold, and 1000-fold. Libraries were constructed using the DNA & RNA co-construction library kit (NadPrep® RNA & DNA co-construction module, catalog number: 1002411, NadPrep (Nanjing) Biotechnology Co., Ltd.). Hybridization was then performed using probe pools covering 100 bp and 500 bp of the target, respectively, using the μCaler® Hybrid Capture Reagents v2 hybridization capture kit (catalog number: 1005102). Sequencing was performed to analyze the number of pathogen sequences detected in different samples. The results showed that using probes designed with a 500 bp target region resulted in a higher number of pathogen sequences detected. From a theoretical perspective, a longer target region generally leads to higher pathogen detection sensitivity, but also higher probe synthesis costs. Therefore, considering the balance between system detection sensitivity and control costs, the system was designed with a target length of 500 bp. The results are shown in Table 6, comparing the pathogen detection performance of different targets; the 500 bp target demonstrated higher detection sensitivity.

[0093] Table 6

[0094]

[0095] The novel coronavirus genome is 29.9 kb in length, so the novel coronavirus genome sequence was selected as the target region; for influenza A virus, the HA and NA genes were selected, with a total length of 1 kb; the target regions of drug resistance genes were selected with a length of 150-200 bp.

[0096] In the design of specific pathogen capture probes, compared with the equal-length probes in traditional hybridization capture experiments, all probes in this scheme are designed with unequal lengths based on the differences in the target region sequence, with probe lengths ranging from 30 to 100 nt. The purpose is to avoid cross-reactions between sequences by dynamically adjusting the probe length, achieving high specificity and consistent hybridization temperature, and shortening the reaction time. The core principle is that the probe length is not fixed but adaptively optimized based on the GC content of the target sequence. Longer probes enhance binding stability, while shorter probes improve discrimination, thus precisely balancing sensitivity and specificity. Simultaneously, adjacent probes bind complementary conjugated sequences, improving reaction efficiency and shortening the hybridization capture time to 30 minutes.

[0097] Taking the Enterococcus faecalis / Serratia marcescens / Escherichia coli / Staphylococcus aureus probe system as an example, compared with traditional probes, its detection sensitivity is significantly improved. Traditional hybridization uses a 2-hour hybridization time, while this system uses a 30-minute hybridization time. The detection sensitivity of this system is significantly higher than that of the traditional 2-hour hybridization. See details below. Figure 2A , Figure 2B , Figure 2C and Figure 2D As shown.

[0098] The target-specific sequences of the probes in the probe pool finally obtained by the above method are shown in SEQ ID No. 1 to SEQ ID No. 600. Among them, bacterial and fungal probes are shown in SEQ ID No. 1 to SEQ ID No. 130; viral probes are shown in SEQ ID No. 131 to SEQ ID No. 425; and drug resistance gene probes are shown in SEQ ID No. 426 to SEQ ID No. 600.

[0099] The 5' end of the target-specific sequence above is connected to the 5' end conjugate sequence A, which is GTCAGTACCG as shown in SEQ ID No. 601, and the 3' end is connected to the 3' end conjugate sequence B, which is CAGTCATGGC as shown in SEQ ID No. 602.

[0100] Example 2: Detection of respiratory multi-pathogen standards using the method provided by the present invention.

[0101] The flowchart of the method for joint detection of multiple pathogens and drug resistance genes based on liquid phase hybridization capture technology of this invention is as follows: Figure 3 As shown, nucleic acid extraction was mainly performed using a DNA and RNA co-extraction kit, and library construction was carried out using a DNA and RNA co-library construction kit (NadPrep® RNA & DNA co-library module, catalog number: 1002411, NadPrep (Nanjing) Biotechnology Co., Ltd.). Hybridization and capture were performed using a specific probe pool, followed by sequencing. The resulting data were analyzed using a specially developed data analysis system, and a results report was generated. The overall workflow achieved simultaneous DNA / RNA pathogen co-detection, precise identification of pathogen subtypes, and whole-genome tracing and drug resistance monitoring, providing strong support for precision treatment, epidemiological research, and epidemic prevention and control. Specific experimental steps are as follows.

[0102] I. Sample Nucleic Acid Preparation:

[0103] The corresponding pathogens and their concentrations were selected and mixed according to the method in Table 7 (where the concentration of human cells incorporated into each gradient was 2 × 10⁻⁶). 5(cell / mL) Simultaneously prepare a blank control (NC) (using an equal volume of PBS buffer, free of human and pathogenic factors). Then, extract according to the corresponding requirements and steps in the nucleic acid extraction kit. The final elution volume is recommended to be 20–50 μL. A universal DNA / RNA co-extraction kit (Tiangen Biotech Co., Ltd., catalog number: TR202) is recommended for nucleic acid extraction. If the extracted pathogenic nucleic acid is not to be used in subsequent experiments, it should be stored at -80℃ and repeated freeze-thaw cycles should be avoided. Before use, it should be thawed on ice or at 4℃.

[0104] Table 7

[0105]

[0106] II. Library Preparation:

[0107] Follow the requirements and steps of the NadPrep® RNA & DNA co-construction library module to construct the library (catalog number: 1002411). The operation steps are as follows:

[0108] 1. RNA denaturation and binding of random primers

[0109] 1) Remove the Random Primer and place it on ice to thaw naturally. Mix well and then centrifuge briefly for later use.

[0110] 2) Prepare the reaction mixture in a 0.2 mL PCR tube placed on ice, according to the table below:

[0111] Table 8

[0112]

[0113] Note: If the RNA is less than 12 μL, it can be supplemented to 12 μL with Nuclease Free Water.

[0114] 3) Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the PCR tube.

[0115] 4) Place the PCR tube into the PCR instrument and start the following program:

[0116] Table 9

[0117]

[0118] Note: During the program, the hot lid should be set to 75℃. After the reaction is completed at 70℃ for 5 minutes, immediately place the container on ice for 2-3 minutes to prevent refolding.

[0119] 2. One-strand synthesis of cDNA

[0120] 1) Take out the 1st Co-Strand Synth. Buffer and the 1st Co-Strand Synth. Enzyme and place them on ice to melt naturally. Mix them evenly and centrifuge briefly for later use.

[0121] 2) Prepare the reaction mixture in a 0.2 mL PCR tube placed on ice, according to the table below:

[0122] Table 10

[0123]

[0124] 3) Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the PCR tube.

[0125] 4) Place the PCR tube into the PCR instrument and start the following program:

[0126] Table 11

[0127]

[0128] Note: The hot cap is set to 90℃ during program execution.

[0129] 5) Store any unused reagents at -25 to -15°C.

[0130] 3. cDNA Two-Strand Synthesis

[0131] 1) Take out the 2nd Co-Strand Synth. Buffer and 2nd Co-Strand Synth. Enzyme and place them on ice to melt naturally. Mix them evenly and centrifuge briefly for later use.

[0132] 2) Prepare the reaction mixture in a 0.2 mL PCR tube placed on ice, according to the table below:

[0133] Table 12

[0134]

[0135] 3) Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the PCR tube.

[0136] 4) Start the following reaction program on the PCR instrument, and place the reaction tube into the PCR instrument when the temperature stabilizes at 16℃.

[0137] Table 13

[0138]

[0139] 4. Fragmentation, end-of-pipe repair, and A addition

[0140] 1) Take out the Co-FERA Buffer and melt it at room temperature. Mix it evenly and place it on ice for later use.

[0141] 2) Take out the Co-FERA Enzyme and place it on ice to melt naturally. Mix it evenly and centrifuge briefly for later use.

[0142] 3) Prepare the reaction system in a 0.2 mL PCR tube placed on ice, according to the table below:

[0143] Table 14

[0144]

[0145] 4) Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the PCR tube.

[0146] 5) Start the following reaction program on the PCR instrument, and place the reaction tube into the PCR instrument once the temperature stabilizes at 20℃:

[0147] Table 15

[0148]

[0149] Note: The hot cap is set to 70°C when this program is running.

[0150] Table 16

[0151]

[0152] 6) Store unused reagents at -25 to -15°C.

[0153] 5. Connector connection

[0154] 1) Take out the Ligation Buffer and melt it at room temperature. Mix it well and place it on ice for later use.

[0155] Note: Ligation Buffer is very viscous; pipette aspiration and dispensing should be slow and steady to ensure accurate volume.

[0156] 2) Remove the DNA ligase and place it on ice to thaw naturally. Mix well and centrifuge briefly for later use.

[0157] 3) Remove the PCR reaction tube from the PCR instrument in step two and place it on ice. Prepare the reaction system according to the table below:

[0158] Table 17

[0159]

[0160] Note: 1. To prevent adapter self-ligation, the NadPrep® Universal Stubby Adapter should be added to the bottom of the PCR tube in step two before adding Ligation Buffer and DNA Ligase. 2. When performing multiple sample operations, to prevent adapter self-ligation due to slow operation, a mixture of Ligation Buffer and DNA Ligase should be pre-prepared in the correct proportions.

[0161] 4) Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the PCR tube.

[0162] 5) Start the following program on the PCR instrument, and place the reaction tube into the PCR instrument when the temperature stabilizes at 20℃.

[0163] Note: This procedure does not require a heated lid.

[0164] Table 18

[0165]

[0166] 6) Store any unused reagents at -25 to -15°C.

[0167] 6. Purification of ligation products

[0168] 1) Remove NadPrep® SP Beads in advance, vortex mix, and equilibrate at room temperature for 30 minutes before use.

[0169] 2) Add 40 μL of NadPrep® SP Beads to the product from the ligation reaction in step 3, mix well, and incubate at 25°C for 5–10 min.

[0170] 3) After briefly centrifuging the PCR tube, place it on a magnetic rack for 5 minutes until the liquid is completely clear. Use a pipette to remove and discard the supernatant.

[0171] Note: It must be clearly stated that the placement time needs to be adjusted for different brands of magnetic racks. The brand used in this example is: Mich Scientific, part number: Magpow-16-0.2, placement time: 5 min.

[0172] 4) Slowly add 150 µL of 80% ethanol along the side wall of the PCR tube, being careful not to disturb the magnetic beads. Let it stand for 30 seconds, then use a pipette to remove and discard the supernatant.

[0173] 5) Repeat step 4) once.

[0174] 6) After briefly centrifuging the PCR tube, place it on a magnetic rack and use a 10 µL pipette tip to remove a small amount of residual ethanol, being careful not to pick up the magnetic beads.

[0175] 7) Open the PCR tube cap and let it stand at room temperature for about 5 minutes until the ethanol has completely evaporated.

[0176] Note: Do not over-dry, otherwise the yield will be reduced.

[0177] 8) Remove the PCR tube, add 20 µL of Nuclease Free Water to the PCR tube, and perform PCR amplification with magnetic beads.

[0178] 7. PCR amplification

[0179] 1) Take out 2× HiFi PCR Master Mix and NadPrep® Universal UDI-Index PrimerMix, place them on ice to thaw naturally, mix well, and centrifuge briefly for later use.

[0180] 2) Prepare the reaction mixture in a 0.2 mL PCR tube placed on ice (add the following ingredients in order from top to bottom):

[0181] Table 19

[0182]

[0183] 3) Place the PCR tube into the PCR instrument and start the following program:

[0184] Table 20

[0185]

[0186] Note: The temperature of the hot cap is stable at 105℃ during program operation.

[0187] Table 21

[0188]

[0189] 4) Store any unused reagents at 25-15℃.

[0190] 8. Amplification library purification

[0191] 1) Add 50 µL of NadPrep® SP Beads to the PCR tube containing the amplification reaction product from step 5, mix well, and incubate at 25°C for 5–10 min.

[0192] 2) After briefly centrifuging the PCR tube, place it on a magnetic rack for 5 minutes until the liquid is completely clear. Use a pipette to remove and discard the supernatant.

[0193] Note: It must be fully clarified that the placement time may vary depending on the brand of the magnetic rack.

[0194] 3) Slowly add 150 µL of 80% ethanol along the side wall of the PCR tube, being careful not to disturb the magnetic beads. Let it stand for 30 seconds, then use a pipette to remove and discard the supernatant.

[0195] 4) Repeat step 3) once.

[0196] 5) After the PCR tubes are briefly centrifuged, place them on a magnetic rack and use a 10 µL pipette tip to remove any remaining ethanol, being careful not to pick up the magnetic beads.

[0197] 6) Open the PCR tube cap and let it stand at room temperature for about 5 minutes until the ethanol has completely evaporated.

[0198] Note: Do not over-dry, otherwise the yield will be reduced.

[0199] 7) Remove the PCR tube, add 20 µL of TE Solution to the PCR tube, use a pipette to suspend the magnetic beads evenly, and incubate at 25°C for 2 min.

[0200] 8) After briefly centrifuging the PCR tube, place it on a magnetic rack for 2 minutes until the liquid is completely clear. Carefully transfer the supernatant to a new 0.2 mL PCR tube for storage using a pipette, being careful not to aspirate the magnetic beads.

[0201] III. Hybrid Capture

[0202] Hybrid capture was performed using the μCaler® Hybrid Capture Reagents v2 Hybrid Capture Kit (catalog number: 1005102), as follows:

[0203] Table 22

[0204]

[0205] Note: If μHyb #1 and Wash Buffer A Pro are to be aliquoted for use / storage, the original tubes / bottles must be heated until the crystals are completely dissolved before aliquoting. Do not aliquot directly. In addition, ensure that all reagent components are fully dissolved and vortexed before use.

[0206] 1. Library hybridization

[0207] 1) Prepare the hybridization reaction system according to the table below:

[0208] Table 23

[0209]

[0210] Note: To maintain library complexity, it is recommended that ≥50% of the total volume of each constructed library be used for hybridization. If the total library volume is >18μL, library concentration can be performed. The number of pre-libraries contained in the total library can be selected as 1, 2, 3, 4, 5, or 6. The recommended input amount for hybridization capture for actual clinical sample library yield is shown in the table below.

[0211] Table 24

[0212]

[0213] 2) Vortex the hybridization reaction mixture for more than 10 seconds, then centrifuge briefly and collect the reaction mixture at the bottom of the PCR tube (avoid generating air bubbles).

[0214] 3) Place the PCR tube into the PCR instrument and start the following reaction program:

[0215] Table 25

[0216]

[0217] Note: Extending the hybridization time may reduce the reaction volume due to evaporation; it is recommended to perform an evaporation test beforehand. Refer to Step 2, Library Capture and Elution, for the capture procedure. For specific temperature selection in the elution procedure, refer to Step 2, Library Capture and Elution.

[0218] Note: The hot cap temperature in the hybridization program is set to 105℃. With 20 minutes remaining in the hybridization program, library capture and elution (magnetic bead cleaning) can be performed. After cleaning, the magnetic beads should be left at room temperature.

[0219] 2. Library capture and elution

[0220] Magnetic bead cleaning

[0221] 1) Vortex the Streptavidin Beads for 15 seconds to ensure complete mixing.

[0222] 2) Take n×25μL of Streptavidin Beads and mix and wash them in a 0.2 mL centrifuge tube (n is the total number of capture reactions, and n≤5).

[0223] Note: Place Wash Buffer A Pro back in a 50-60°C environment to heat.

[0224] 3) Place the Streptavidin Beads on a magnetic rack and let stand for about 2 minutes until the liquid is completely clear. Then discard the supernatant using a pipette.

[0225] 4) Remove the centrifuge tube from the magnetic rack, add 150 μL of preheated Wash Buffer A Pro, and gently blow and aspirate to mix more than 10 times.

[0226] 5) Place the centrifuge tube on a magnetic rack and let it stand for about 2 minutes until the liquid is completely clear. Then discard the supernatant using a pipette.

[0227] 6) Repeat steps 4) and 5) once.

[0228] Note: When n > 5, the cleaning must be performed in multiple tubes.

[0229] 7) After briefly centrifuging the centrifuge tubes, place them on a magnetic rack and let them stand for 10 seconds. Use a 10 μL pipette tip to remove all the WashBuffer A Pro from the bottom of the tubes.

[0230] 8) Take n×9 μL μHyb #1 to resuspend the Streptavidin Beads and gently blow-wash and mix more than 10 times.

[0231] magnetic bead capture

[0232] 1) After the hybridization reaction, the process enters the capture phase to maintain the operation of the PCR instrument.

[0233] 2) While the PCR tubes are in the PCR instrument, take 8 μL of resuspended Streptavidin Beads and immediately add them to each hybridization reaction solution. Gently pipette and mix at least 10 times.

[0234] 3) Incubate at 60℃ for 10 min.

[0235] 4) After incubation, remove the PCR tube from the PCR instrument and place it on a magnetic rack for 2 minutes until the liquid is completely clear. Use a pipette to discard the supernatant (try to remove the supernatant as completely as possible).

[0236] Washout

[0237] 1) Remove the PCR tube from the magnetic rack, add 150 μL of preheated Wash Buffer A Pro, and gently pipette to mix at least 10 times (avoid generating air bubbles).

[0238] 2) Place the PCR tube on a magnetic rack and let it stand for 2 minutes until the liquid is completely clear. Then discard the supernatant using a pipette.

[0239] 3) Remove the PCR tube from the magnetic rack, add 100 μL of preheated Wash Buffer A Pro, gently pipette and mix more than 10 times, and transfer the reaction solution to a new PCR tube.

[0240] Note: Try to avoid generating air bubbles in this step, as air bubbles coming into contact with the tube cap may reduce the hit rate.

[0241] 4) Place the PCR tube into the PCR instrument and incubate at 60°C for 3 min.

[0242] 5) After incubation, remove the PCR tube from the PCR instrument and place it on a magnetic rack for 2 minutes until the liquid is completely clear. Then, use a pipette to discard the supernatant.

[0243] 6) Add 150 μL of room temperature Wash Buffer B to the PCR tube and gently pipette to mix more than 10 times.

[0244] 7) Place the PCR tube on a magnetic rack and let it stand for 2 minutes until the liquid is completely clear. Then discard the supernatant using a pipette.

[0245] 8) After briefly centrifuging the PCR tube, place it on a magnetic rack and use a 10 µL pipette tip to remove a small amount of residual liquid, being careful not to pick up the magnetic beads.

[0246] 9) Add 22.5μL of Nuclease Free Water and gently rinse the resuspended magnetic beads more than 10 times.

[0247] Optional step: Transferring all resuspended magnetic beads to a new PCR tube can reduce non-specific capture and improve the hit rate.

[0248] 3. PCR amplification of hybridization capture library

[0249] 1) Take out 2× PCR Master Mix and Primer Mix and thaw them naturally on ice. Use a pipette or vortex mixer to gently mix them evenly and then centrifuge briefly for later use.

[0250] 2) Prepare the reaction system in centrifuge tubes placed on ice according to the system in the table below.

[0251] Table 26

[0252]

[0253] 3) Place the PCR tube into the PCR instrument and start the following program, as shown in the table below.

[0254] Table 27

[0255]

[0256] Note: Immediately after amplification, use Qubit to detect the concentration. A concentration >2 ng / µL is acceptable; do not over-amplify.

[0257] 4. Library purification and quantification

[0258] 1) After PCR amplification is complete, remove the PCR tube and place it on a magnetic rack for 2 minutes to allow the supernatant to become completely clear. Then, use a pipette to transfer the supernatant to a new centrifuge tube.

[0259] 2) Add 50 µL of NanoPrep SP Beads to the PCR tube and mix thoroughly using a pipette or vortex mixer. Incubate at 25°C for 5 min.

[0260] 3) After briefly centrifuging the PCR tube, place it on a magnetic rack for 5 minutes until the supernatant is completely clear. Use a pipette to remove and discard the supernatant.

[0261] 4) Slowly add 150 µL of 80% ethanol along the side wall of the PCR tube, being careful not to disturb the magnetic beads. Let it stand for 30 seconds, then use a pipette to remove the supernatant.

[0262] 5) Repeat step 4) once.

[0263] 6) Briefly centrifuge the PCR tubes, place them on a magnetic rack, and use a 10 µL pipette tip to remove a small amount of residual ethanol, being careful not to aspirate the magnetic beads.

[0264] 7) Place the PCR tube back on the magnetic rack and let it stand at room temperature for 5 minutes until the ethanol has completely evaporated (do not dry it too much, as this will damage the DNA and make it difficult to reconstitute).

[0265] 8) Add 15 µL of TE Solution to the PCR tube, use a pipette to suspend the magnetic beads evenly, and incubate at 25°C for 2 min.

[0266] 9) After briefly centrifuging the PCR tube, place it on a magnetic rack for 2 minutes until the supernatant is completely clear. Carefully transfer the supernatant to a new centrifuge tube for storage using a pipette, being careful not to aspirate the magnetic beads.

[0267] 10) Use Qubit to quantify the library at a concentration >2 ng / µL.

[0268] IV. Sequencing

[0269] Each library has no special requirements depending on the purpose of the detection; a size of ≥0.5G is recommended.

[0270] V. Test Results

[0271] 1. Sequencing data quality control results:

[0272] like Figure 4As shown, the Q30 of T1–T3 and T5 were all ≥90%, indicating good sequencing quality; the average percentage of valid reads was ≥99%, and the average percentage of human samples was 67%, indicating high data utilization. The Q30 of the NC sample was 81.07%, with 9437 sequencing reads. Data analysis revealed no microorganisms, indicating a clean background signal during the detection process and that the specific probe pool provided by this invention does not undergo significant cross-hybridization with non-target pathogens (especially closely related species with high genomic similarity) or human genomes.

[0273] 2. Pathogen detection results:

[0274] As shown in Table 28, Aspergillus fumigatus and Acinetobacter baumannii in group C1 of T5 may not have been detected because the number of pathogens in this group was small and the concentration was too low, resulting in the inability to extract nucleic acids. The others were detected normally, indicating that the present invention has excellent ability to detect multiple pathogens.

[0275] Table 28

[0276]

[0277] Note: +: indicates normal detection; -: indicates no detection; blank: indicates no pathogen contamination.

[0278] 3. Calculation methods and results of positive concordance rates for different samples and pathogens:

[0279] The positive concordance rate of a sample = number of pathogens detected in the sample / total number of pathogens originally present in the sample × 100%. For example, in group C5 of T5, a total of 5 pathogens were mixed in and all 5 were detected normally. Therefore, the positive concordance rate = 5 / 5 × 100% = 100%.

[0280] The positive concordance rate for pathogens = number of concentration gradients detected / total number of concentration gradients for pathogens × 100%. For example, if Enterococcus faecalis T1 is detected in all 5 concentration gradients, then the positive concordance rate = 5 / 5 × 100% = 100%.

[0281] like Figure 5 and Figure 6 As shown, the positive concordance rates for T1 to T3 at different concentration gradients were all 100%. For sample T5, except for C1, the positive concordance rates for the other four gradients were all 100%. Analysis of the five concentration gradients (C1-C5) for Aspergillus fumigatus and Acinetobacter baumannii revealed that the concentration of C1 might have been too low, resulting in loss of nucleic acid during extraction and failure to detect it. The detection results for different pathogens in each sample group showed that, except for sample T5 where the positive concordance rate for Aspergillus fumigatus and Acinetobacter baumannii was 80%, the positive concordance rate for all other pathogens was 100%, indicating that this invention has high detection sensitivity.

[0282] Example 3: Detecting the novel coronavirus and tracking its mutations using the method provided by this invention.

[0283] I. Nucleic Acid Extraction from Samples: Four clinical samples (A1–A4) of novel coronavirus infection were provided by Beijing You'an Hospital, affiliated with Capital Medical University. Nucleic acid was extracted from the clinical samples according to the extraction method described in Example 2.

[0284] II. Library Construction: Proceed as described in Example 2.

[0285] Hybrid capture: Performed as in Example 2.

[0286] IV. Sequencing: Each library has no special requirements depending on the purpose of the test; ≥0.5G is recommended.

[0287] V. Experimental Results: (e.g.) Figure 7A As for Figure 7D As shown. Figure 7A The target rate in samples A1 to A4 was ≥90%; Figure 7B In the range of 0.2×Mean ≥ 98%, 0.5×Mean ≥ 89%, the average sequencing depth after deduplication is 142. Figure 7C As shown, the sequencing data from different samples exhibit good homogeneity and high probe specificity; simultaneously, the whole genome coverage of the novel coronavirus in strains A1–A4 is very high, and all strains can be successfully assembled, spliced, and annotated to their corresponding strains, such as… Figure 7D As shown, this invention enables whole-genome detection and mutation tracking of the novel coronavirus.

[0288] Example 4: Detection of Mycobacterium tuberculosis drug resistance genes using the method provided by this invention.

[0289] I. Nucleic Acid Extraction from Samples: Three clinical samples (R1-R3) of tuberculous branch infection were provided by Beijing You'an Hospital, Capital Medical University. Nucleic acid was extracted from the clinical samples according to the extraction method described in Example 2.

[0290] II. Library Construction: Proceed as described in Example 2.

[0291] III. Hybrid capture: Performed in accordance with the method of Example 2.

[0292] IV. Sequencing: Each library has no special requirements depending on the purpose of the test; ≥0.5G is recommended.

[0293] V. Experimental Results

[0294] 1. Pathogen detection results:

[0295] As shown in Table 29, Mycobacterium tuberculosis and Mycobacterium jirovecii were detected in high reads in samples R1 to R3. Streptococcus pneumoniae was also detected in R2, indicating that the present invention can achieve co-detection of multiple pathogens.

[0296] Table 29

[0297]

[0298] 2. Results of drug resistance gene testing:

[0299] As shown in Table 30, R1 to R3 jointly detected Erm(37), rpoB2, and rpoB, with Erm(37) coverage ≥90%, rpoB2 coverage ≥47%, and rpoB coverage ≥17%. The sequencing depth of the drug resistance gene corresponding to each sample was basically consistent with the detection results of Mycobacterium tuberculosis in each sample, indicating that the present invention can normally detect the relevant drug resistance genes of Mycobacterium tuberculosis and realize the co-detection of multiple pathogens and drug resistance genes.

[0300] Table 30

[0301]

[0302] Example 5: Detection of different types of clinical samples using the method provided by the present invention.

[0303] I. Nucleic acid extraction from samples: Three clinical samples (L1-L3) of different types of respiratory tract infections, namely one pharyngeal swab, one bronchoalveolar lavage fluid and one sputum, were provided by the nucleic acid sequencing platform of Beijing You'an Hospital affiliated to Capital Medical University. Nucleic acid was extracted according to the extraction method in Example 1.

[0304] II. Library Construction: Proceed as described in Example 2.

[0305] III. Hybrid capture: Performed in accordance with the method of Example 2.

[0306] IV. Sequencing: Each library has no special requirements depending on the purpose of the test; ≥0.5G is recommended.

[0307] V. Experimental Results

[0308] 1. Sequencing data quality control results: As shown in Table 31, the Q30 of L1 to L3 is ≥90%, indicating good data quality; the percentage of valid reads is ≥99%, and the percentage of human sources is ≤50%, indicating high data utilization.

[0309] Table 31

[0310]

[0311] 2. Pathogen Detection Results: As shown in Table 32, the main pathogen detected in sample L1 was influenza A virus H3N2, the main pathogens detected in L2 were novel coronavirus and Klebsiella pneumoniae, and the main pathogens detected in L3 were Enterococcus faecalis and Aspergillus fumigatus. These three sample types cover bacteria, fungi, and viruses, indicating that this invention can achieve multi-pathogen co-detection and is applicable to different sample types in clinical settings.

[0312] Table 32

[0313]

[0314] The experimental data above also show that the present invention can avoid interference from host nucleic acids and exhibits excellent detection performance even under high host background conditions (such as sputum with a high concentration of human cells). The stability and detection capability of the method were verified in different types of clinical samples with varying processing conditions (such as throat swabs, bronchoalveolar lavage fluid, and sputum).

[0315] Example 6: Detection of clinical samples infected with different viruses using the method provided by this invention and mNGS.

[0316] I. Nucleic acid extraction from samples: Four clinical samples infected with different viruses (S1-S4), of which S1-S2 were infected with the novel coronavirus and S3-S4 were infected with influenza A virus H1N1. All samples were provided by the nucleic acid sequencing platform of Beijing You'an Hospital affiliated to Capital Medical University and nucleic acid was extracted according to the extraction method in Example 1.

[0317] II. Library Construction: The process was performed according to Example 2. mNGS was conducted following the operating procedures of the nucleic acid sequencing platform at Beijing You'an Hospital, affiliated with Capital Medical University.

[0318] III. Hybrid capture: Performed in accordance with the method of Example 2.

[0319] IV. Sequencing: Each library has no special requirements depending on the purpose of the test; ≥0.5G is recommended.

[0320] V. Experimental Results: As shown in Table 33, the detection results of the present invention are completely consistent with mNGS, and even the number of reads detected in pathogens is higher than that of mNGS.

[0321] Table 33

[0322]

[0323] Note: +: indicates normal detection; (): indicates the number of reads for the corresponding pathogen detected.

[0324] Example 7: Detection of cultured drug-resistant bacteria using the method provided by the present invention.

[0325] I. Nucleic acid extraction from samples: The nucleic acid sequencing platform of Beijing You'an Hospital, affiliated with Capital Medical University, was used to extract nucleic acid from the three cultured drug-resistant bacteria (M1-M3). All samples had undergone phenotypic drug resistance identification and nucleic acid extraction was performed according to the extraction method in Example 2.

[0326] II. Library Construction: Proceed as described in Example 2.

[0327] III. Hybrid capture: Performed in accordance with the method of Example 2.

[0328] IV. Sequencing: Each library has no special requirements depending on the purpose of the test; ≥0.5G is recommended.

[0329] V. Experimental Results: As shown in Table 34, the detection results of this invention are completely consistent with the results of phenotypic drug susceptibility testing, indicating that this invention has good drug resistance gene detection capabilities.

[0330] Table 34

[0331]

[0332] Example 8: Detection of different subtypes of adenovirus using the method provided by the present invention.

[0333] I. Nucleic acid extraction from samples: Clinical samples were all provided by the nucleic acid sequencing platform of Beijing You'an Hospital, affiliated with Capital Medical University, and nucleic acid was extracted according to the extraction method in Example 2.

[0334] II. Library Construction: Proceed as described in Example 2.

[0335] III. Hybrid capture: Performed in accordance with the method of Example 2.

[0336] IV. Sequencing: Each library has no special requirements depending on the purpose of the test; ≥0.5G is recommended.

[0337] V. Experimental Results: Detection results: As shown in Table 35, this system can detect different subtypes of adenovirus.

[0338] Table 35

[0339]

Claims

1. A liquid hybridization probe pool for the combined detection of multiple pathogens and drug resistance genes, characterized in that The probes are designed according to the target pathogen and the target region of the drug resistance gene; the probes are unequal length probes with a length range of 30-100 nt; the probes include a target-specific sequence complementary to the target region of the target pathogen and a conjugate sequence that can be complementary to the adjacent probe after hybridization; the 5' and 3' ends of the probe are modified with conjugate sequences A and B, respectively; the 5'-end conjugate sequence A of one probe is reverse complementary to the 3'-end conjugate sequence B of another probe, so that the adjacent probes can specifically bind through end pairing; the 5'-end conjugate sequence A is shown as SEQ ID No. 601, and the 3'-end conjugate sequence B is shown as SEQ ID No. 602; the target-specific sequence is shown as SEQ ID No. 1 to SEQ ID No.

600.

2. The liquid hybridization probe pool for combined detection of multiple pathogens and drug resistance genes according to claim 1, wherein: the target pathogen is one or more of the following: novel coronavirus, influenza A virus, adenovirus, Mycobacterium tuberculosis, Aspergillus fumigatus, Enterococcus faecium, Streptococcus pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Listeria monocytogenes, Enterobacter cloacae, Pseudomonas aeruginosa, Neisseria meningitidis, Klebsiella pneumoniae, Acinetobacter baumannii, Chlamydia pneumoniae, respiratory syncytial virus, human parainfluenza virus, and influenza B virus.

3. The liquid hybridization probe pool for combined detection of multiple pathogens and drug resistance genes according to claim 1, wherein: The drug resistance gene is Erm (37) , rpoB , rpoB2 gene of Mycobacterium tuberculosis, the TEM-19 , OXA-83 gene of Acinetobacter baumannii, and the kpnF, KPC-2, acrR, ompk37 gene of Klebsiella pneumoniae.

4. A method for designing a pool of liquid hybridization probes according to any one of claims 1 to 3, characterized in that comprising the following steps: (1) input initial sequence information and design parameters: the initial sequence information includes total sequence information and target sequence information; the design parameters are: according to the GC content of the target sequence, the hybridization temperature range of the probe and the target sequence, the length range of the probe sequence and the length range of the conjugate sequence are set, and the probe is designed with unequal length; The formula for calculating the Tm value of the DNA sequence is as follows: Tm=81.5+0.41\times(\%G+\%C)-\frac{675}{N}+16.6\times\log_{10}(0.05); (2) subsequence extraction and statistics: all sub-sequences with a length of k are extracted from the positive strand and complementary strand sequences of the total sequence, and the number of occurrences of each sub-sequence is counted to provide data support for the selection of conjugate sequences in the subsequent step; (3) conjugate sequence design: according to the design parameters set in step (1), conjugate sequences with a length of k are selected; The conjugate sequence selection needs to meet the following conditions: the hybridization temperature of the selected conjugate sequence is lower than the hybridization temperature of the probe and the target sequence; the number of occurrences of the selected conjugate sequence k in the total sequence is less than 10% of the average value. (4) Target-specific sequence: select each target sequence i in turn, starting from i = 1; select the target-specific sequence from the nth base of each target sequence, starting from n = 1, to ensure that the probe has high specificity in binding with the nucleic acid target sequence; (5) Probe sequence construction: add the conjugate sequence screened in step (3) to the 5' end and 3' end of the target-specific sequence, respectively, to construct the liquid hybridization probe pool.

5. The design method of the liquid hybridization probe pool according to claim 4, characterized in that: The total sequence information is extracted from all possible sequences contained in the pre-capture library; the target sequence information is the sequence to be captured, and avoids low-specificity sequences such as repetitive sequences.

6. The liquid hybridization probe pool according to any one of claims 1 to 3 for use in the preparation of the combined detection kit, the tracing kit and / or the typing detection kit for the target pathogen according to claim 2 and / or the drug-resistant gene according to claim 3.

7. A multi-pathogen and drug resistance gene combined detection kit, characterized in that Comprising: (1) The liquid hybridization probe pool according to any one of claims 1 to 3; (2) Liquid hybridization capture reagent; (3) DNA / RNA co-library reagent; (4) Nucleic acid extraction reagent.

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