An enterovirus liquid probe target capture library construction method and kit thereof

Abstract

This invention discloses a liquid-phase probe-targeted capture library construction method and kit for enteroviruses, belonging to the field of virus detection technology. This invention combines targeted enrichment with high-throughput sequencing, featuring broad coverage, high flexibility, high sensitivity, and high specificity, enabling multi-dimensional information output including quantitative, qualitative, and in-depth analysis. Through the probe-targeted capture "enrichment-screening" mechanism, this invention reduces probe redundancy design, minimizes background nucleic acid interference from intestinal flora, exhibits excellent adaptability to complex samples, and eliminates the need for complex sample pretreatment, making it suitable for large-scale epidemiological detection.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to a method and kit for targeted capture and library preparation of enteroviruses using liquid-phase probes. Background Technology

[0002] Enteroviruses are a class of viruses that primarily infect and replicate in the intestines. They belong to the Picornaviridae family, are highly infectious, and can cause a variety of diseases, having a significant impact on human health, especially children's health.

[0003] Common types of enteroviruses include: Poliovirus: This is a well-known enterovirus that can cause poliomyelitis (commonly known as infantile paralysis). The virus mainly attacks the motor nerve cells of the central nervous system, leading to limb paralysis or even paralysis.

[0004] Coxsackieviruses are divided into group A and group B. Group A Coxsackieviruses can cause hand-foot-mouth disease, herpetic pharyngitis, etc.; Group B Coxsackieviruses may cause myocarditis, pericarditis, meningitis, and other diseases.

[0005] Echovirus: also known as enterocytic human orphan virus, which can cause symptoms such as fever, rash, and meningitis, and the clinical manifestations of the diseases it causes are diverse.

[0006] Novel enteroviruses: With the progress of virological research, new enterovirus serotypes are constantly being discovered, such as enterovirus 71 (EV71), which is one of the important pathogens causing hand-foot-mouth disease and is prone to causing severe cases.

[0007] Diseases and clinical manifestations caused by: Enterovirus infection presents with a wide range of clinical manifestations, from asymptomatic infection to severe central nervous system disorders and even death. Common diseases and manifestations are as follows: Hand, foot, and mouth disease (HFMD): Most commonly affects children under 5 years old and is caused by Coxsackievirus A16, EV71, etc. The main symptoms are rashes and blisters on the hands, feet, and mouth, which may be accompanied by fever, sore throat, and other symptoms. A small number of children may develop severe illness, including meningitis, encephalitis, and pulmonary edema.

[0008] Herpetic pharyngitis: mainly caused by Coxsackievirus A, it is characterized by fever, sore throat, grayish-white vesicles on the pharyngeal mucosa with redness around them, and ulcers formed after the vesicles rupture.

[0009] Meningitis and encephalitis: Various enteroviruses can cause central nervous system infections. Patients may experience symptoms such as fever, headache, vomiting, neck stiffness, and altered consciousness. In severe cases, sequelae may remain.

[0010] Myocarditis and pericarditis: Coxsackievirus B is a common pathogen. Patients may experience symptoms such as chest tightness, chest pain, palpitations, and difficulty breathing. In severe cases, it can lead to heart failure.

[0011] Other diseases: It can also cause epidemic chest pain, acute hemorrhagic conjunctivitis, respiratory tract infections, etc.

[0012] In summary, there are many types of enteroviruses, they spread widely, cause a variety of diseases, and require advanced detection techniques.

[0013] Currently, the detection methods generally include RT-qPCR and metagenomic library preparation and sequencing. (1) The RT-qPCR detection method has the advantages of high sensitivity, which can detect viral RNA as low as 10-100 copies / μL, and is suitable for early infection (before the appearance of symptoms) or low viral load samples; high specificity, which can distinguish enteroviruses from other viruses (such as adenoviruses) through specific primers and probes, and can even distinguish different serotypes; rapid and efficient, which only takes 3-6 hours from sample processing to result output, much faster than traditional virus isolation and culture, and can also assess the severity of infection through viral load. However, this method has certain limitations. For example, RT-qPCR detects viral nucleic acid, which may detect inactivated viruses. It cannot distinguish whether the detected virus is live or dead. It is difficult to compare its conserved sequence. If the virus undergoes functional group mutations that cause changes in the conserved sequence, false negatives may occur. The technical threshold is relatively high. Aerosol contamination of amplification products may lead to false positives.

[0014] (2) Metagenomic library construction and sequencing method. This method has unbiased detection and can cover known and unknown enteroviruses. It can simultaneously detect the nucleic acids of all microorganisms such as multiple pathogens, bacteria, fungi, and parasites, and identify all potential pathogens and their relative abundance at one time. It provides rich genomic information and supports in-depth analysis. This method can not only detect enteroviruses, but also obtain the complete or partial genome sequence of the virus. However, this detection method is expensive. Metagenomic library construction and sequencing covers the whole genome sequence of the microorganisms used in the sample, including a large number of non-target species, host contamination DNA and environmental background nucleic acids, which requires the generation of massive sequencing data to meet the analysis needs. The sequencing depth of the target region of this method is insufficient, and the detection sensitivity of low abundance targets is low. For low abundance target substances / genes in clinical testing, microbial tracing and other scenarios, their sequences may be submerged by the sequences of high abundance species, resulting in insufficient sequencing depth of the target region and failure to reach the threshold for reliable detection. Furthermore, metagenomic sequencing is difficult to analyze for samples with high numerical complexity. It cannot actively exclude host sequences, resulting in a large amount of sequencing resources being wasted on non-target host nucleic acids. This further compresses the sequencing depth of the target microorganism, and may even lead to the target not being detected. For sequences of low abundance or rare species, they may not be able to be assembled into complete fragments due to "insufficient coverage", resulting in only fragmented short-read sequences, which makes it difficult to achieve species identification, gene function annotation or genome assembly. Summary of the Invention

[0015] To address the shortcomings of the aforementioned technologies, the probe-targeted capture technology proposed in this invention essentially combines targeted enrichment with high-throughput sequencing, cleverly compensating for the "narrow coverage" of qPCR and the "low sensitivity and high redundancy" of metagenomic sequencing, thus forming a unique technological advantage.

[0016] To achieve the above technical objectives, this invention provides a method for targeted capture and library construction of enteroviruses using liquid-phase probes, comprising the following steps: S1. Sample processing and double-stranded cDNA synthesis: Enterovirus RNA was extracted from the sample using a viral RNA extraction kit, the RNA was fragmented, the fragmented RNA was reverse transcribed into first-strand cDNA, the first-strand cDNA was synthesized into enterovirus double-stranded cDNA and purified to a concentration ≥1 ng / μL; S2. Constructing the initial cDNA library: The double-stranded cDNA is fragmented to a length of 200-250 bp, and the fragments are purified by end repair, A-tailing, adapter ligation, and PCR amplification to obtain the initial library; S3. Probe and library hybridization capture: The initial library is mixed with the probe pool and hybridization blocking system, denatured, and hybridized at 60-65℃ for 1-4 hours. The hybridization product is captured using streptavidin magnetic beads, washed to remove non-specific bindings, and the virus-enriched library is obtained by elution. S4. Post-capture amplification and purification: The enriched library was amplified and purified by PCR. Obtain viral targeted sequencing libraries suitable for high-throughput sequencing; The probe pool contains a set of oligonucleotide probes capable of specifically binding to conserved regions or coding regions of enterovirus genomes. The nucleotide sequences of the probes are selected from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 30, or variant sequences having at least 90% sequence identity with them.

[0017] The length of a single probe is 100 bp. The sequencing data showed 100% coverage of enterovirus genomes. Further, the enterovirus mentioned in step S1 is one or more of poliovirus, Coxsackievirus A16, echovirus 9, or enterovirus 71.

[0018] Furthermore, the reverse transcription system described in step S1 includes reverse transcriptase, primers, and deoxynucleotide triphosphates; the fragmentation described in step S2 is achieved by enzymatic fragmentation or sonication.

[0019] Furthermore, the mixing ratio of the initial library and the probe pool in step S3 is 500 ng: 4 μL.

[0020] Furthermore, the hybridization blocking system includes a probe blocker, a repeat sequence blocker, an adapter blocker, and a hybridization buffer, wherein the nucleotide sequence of the probe blocker is selected from the sequence shown in SEQ ID NO: 31. Furthermore, the library can be directly used in sequencing platforms such as Illumina and MGI.

[0021] This invention also provides an enterovirus kit, comprising the probe pool, probe preservation solution, hybridization blocking system (Micro Probe Hyb Mixture Plus), streptavidin magnetic beads, and washing buffer. The hybridization blocking system includes universal blockers, repeat sequence blockers (COT DNA), and adapter blockers.

[0022] Furthermore, the kit can detect fecal, blood, cerebrospinal fluid, or pharyngeal swab samples with a sensitivity as low as 10² copies / μL.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Balance between coverage and flexibility: The sequencing data covers the entire viral genome, with 100% coverage of enterovirus genomes. This not only breaks through the limitation of qPCR "one target, one test", but also covers multiple enteroviruses (including common viruses, rare viruses and known variants) at once. It also avoids the blindness of metagenomic sequencing "indiscriminate coverage". It can focus on specific viral groups of clinical or scientific interest (such as only enteroRNA viruses or DNA viruses) through customized probe libraries, which is highly flexible.

[0024] (2) The unity of high sensitivity and high specificity: By enriching probes, viral sequences are "fished out" from massive background nucleic acids, effectively increasing sequencing depth. The detection rate of low-abundance viruses far exceeds that of metagenomic sequencing and is comparable to qPCR. The probe design is based on conserved regions of the virus and has extremely low cross-binding rate with non-target sequences (host, bacteria). Combined with sequence verification of sequencing, host nucleic acid interference is reduced, and the proportion of target pathogens in the library is increased by more than 30%. The specificity is better than metagenomic sequencing. The probe combined with magnetic bead enrichment technology can detect enteroviruses as low as 10² copies / μL. The sensitivity is significantly better than traditional metagenomic methods and avoids the possible primer cross-reaction problem of qPCR.

[0025] (3) Multidimensional information output of “quantitative + qualitative + in-depth analysis”: This combination of “quantitative + sequence information” is something that neither qPCR (without sequence information) nor metagenomic sequencing (low quantitative accuracy) can achieve alone.

[0026] (4) Strong adaptability to complex samples: Intestinal samples (feces, rectal swabs) contain more than 99% host DNA and intestinal flora nucleic acids, which are the main sources of interference in virus detection. Probe-targeted capture directly reduces the proportion of background nucleic acids through an "enrichment-screening" mechanism, and its adaptability to such complex samples is far superior to metagenomic sequencing, and it does not require complex sample pretreatment.

[0027] (5) Simple operation and low cost: It integrates enzyme digestion and fragmentation, magnetic bead purification and hybridization capture technology, shortens the library construction time to 8 hours, is compatible with conventional PCR instruments and mainstream sequencing platforms, requires no special equipment, reduces probe redundancy design, reduces the cost of a single reaction by 20%, and is suitable for large-scale epidemic monitoring.

[0028] (6) Clinical samples of enterovirus infection (such as feces, cerebrospinal fluid, and pharyngeal swabs) often present two major problems: Low viral load: The viral nucleic acid copy number may be extremely low in samples from asymptomatic infected individuals or patients in the recovery period (e.g., <10³ copies / g in fecal samples). Strong matrix interference: The sample contains a large amount of host nucleic acid (such as human genomic DNA), microbial nucleic acid and inhibitors (such as polysaccharides and proteins in feces), which interfere with routine PCR detection.

[0029] The probe capture technology of this invention directly enriches enterovirus sequences from a complex background by hybridizing specific probes with the target viral nucleic acid, without relying on PCR pre-amplification (or only requiring low cycle number amplification), and can effectively avoid the problems of "low-load template amplification failure" or "inhibitors causing a decrease in PCR efficiency".

[0030] Compared to traditional RT-PCR or multiplex amplification techniques, its detection sensitivity for low-load samples can be increased by 10-100 times, making it particularly suitable for "zero-case" surveillance of poliovirus (where trace amounts of virus need to be detected to confirm the eradication effect).

[0031] Enteroviruses comprise more than 100 serotypes, and the genomic sequences of different serotypes vary considerably (e.g., the nucleotide differences in the VP1 region can exceed 30%). Traditional methods (such as multiplex PCR) rely on type-specific primers, which can easily lead to missed detections due to primer mismatch with novel serotype sequences.

[0032] Probe capture technology, through the design of hybrid probe libraries covering conserved and hypervariable regions of multiple serotypes (such as probes designed for the conserved 5'UTR region and the hypervariable VP1 region), can simultaneously capture multiple types of enteroviruses. There is no need to design primers separately for each serotype, reducing false negatives due to serotype diversity; For novel recombinant strains that have not yet been discovered (such as Coxsackievirus A group and echovirus recombinants), their sequences can still be captured by conserved region probes, providing a basis for novel identification.

[0033] Enteroviruses are single-stranded positive-sense RNA viruses with a genome length of approximately 7.4-8.3 kb, containing multiple functional regions (such as the 5'UTR, VP1-VP4 structural protein regions, and the 3D polymerase region). Traditional amplicon sequencing often results in incomplete coverage of certain regions (such as the 3'UTR with high GC content) due to variations in primer binding sites or differences in amplification efficiency, affecting the detection of key variants (such as vaccine escape mutations and drug resistance mutations).

[0034] Probe capture technology enables genome-wide probe coverage (e.g., designing probes to cover the entire ORF region) to: Reduce amplification bias: Avoid the loss of certain fragments due to differences in primer efficiency, and the genome coverage can usually reach more than 95% (the average of traditional methods is 70%-80%). Complete analysis of variation patterns: For example, in the differentiation between poliovirus vaccine strains and wild-type strains, characteristic mutations in the 5'UTR and VP1 regions can be accurately detected, avoiding misjudgments caused by fragment deletions.

[0035] In enterovirus infections, co-infection with multiple serotypes (such as the same patient being infected with both Coxsackievirus A16 and Echovirus 9) is not uncommon, and the virus readily produces quasi-species (a group of variants of the same strain) during replication. Traditional methods (such as genotyping PCR) are difficult to use to identify multiple serotypes simultaneously, and the bias of PCR amplification can mask low-frequency variants (such as the 1%-5% quasi-species ratio).

[0036] The advantages of probe capture technology are: Unbiased enrichment: independent of PCR pre-amplification (or low-cycle amplification), it can preserve the original proportion of viral population in the sample and accurately detect each serotype in multiple infections; Low-frequency variant detection: It can capture quasi-species variants as low as 1% (such as antigenic site mutations in the VP1 protein), providing more refined data for studying the evolutionary path and transmission chain of enteroviruses.

[0037] Enterovirus surveillance often requires processing large numbers of samples (such as fecal sample screening in polio eradication programs). Probe capture technology can be automated through magnetic bead methods (such as handling hybridization, washing, and enrichment steps in a liquid workstation), reducing manual intervention. It is also compatible with high-throughput sequencing platforms (such as Illumina NovaSeq), allowing hundreds of samples to be tested in a single experiment, significantly improving the efficiency of large-scale surveillance.

[0038] In summary, probe capture technology, with its advantages of high specificity enrichment, low load detection, complete genome coverage, and unbiased population analysis, is perfectly suited to the characteristics of enteroviruses, which are characterized by "multiple types, rapid mutation, and complex samples," and is irreplaceable, especially in the following scenarios: Monitoring and eradication verification of low viral loads of viruses such as polio; Discovery and identification of novel enteroviruses; Multiple infection analysis in complex samples (such as feces, cerebrospinal fluid); Research on the evolution and transmission chain of viral quasi-species.

[0039] Although it is slightly more expensive than traditional PCR or multiplex amplification techniques, its advantages in detection accuracy and information richness make it an important technical means in molecular diagnosis and scientific research of enteroviruses. Attached Figure Description

[0040] Figure 1 Design diagram for whole-genome capture probes; Figure 2This is a verification graph for the detection limit. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0042] Example 1: Enterovirus probe capture and library construction (1) Probe design: like Figure 1 The diagram shows the design of whole-genome capture probes. Based on conserved regions of various enterovirus genomes (including the 5'UTR conserved region, conserved fragments of VP1-VP4 structural proteins, and the 3D polymerase coding region), multiple biotin-labeled DNA probes were designed. The probes are 100 bp in length and cover the entire viral genome (approximately 7.2-8.5 kb). Reference genome numbers are: poliovirus (GenBank accession number NC_002058.3), Coxsackievirus A16 (GenBank accession number NC_001612.1), Echovirus 9 (GenBank accession number NC_001472.1), and Enterovirus 71 (GenBank accession number NC_002037.1).

[0043] The nucleotide sequences of the obtained probes are shown in SEQ ID NO.1 to SEQ ID NO.30. The nucleotide sequence of the probe blocker is shown in SEQ ID NO.31.

[0044] Target gene screening and coverage strategies: 1. Conserved region selection: The probe design is based on the 5'UTR conserved region and the 3D polymerase coding region, making it suitable for designing probes across multiple viral strains; 2. High-information region selection: The conserved fragments of VP1-VP4 structural proteins (key genes for genotyping) are selected as high-information region target genes to ensure that the probe covers the characteristic sites of all known genotypes; 3. Whole genome coverage: Using the "uniform spacing design method", one probe is designed every 100-150 bp within the core region of the whole genome (7.2-8.5 kb) to avoid overlapping regions (overlap rate ≤30%), while excluding extreme regions in the genome with GC content <30% or >60% to ensure uniform hybridization efficiency.

[0045] The nucleotide sequences of the probe and probe blocker are shown in Table 1.

[0046] Table 1. Nucleotide sequences of probes and probe blockers (2) Sample preparation: Take 0.2g of fecal sample and extract RNA using a viral RNA extraction kit to obtain 30μL of elution buffer containing viral RNA. To improve reverse transcription efficiency, place the extracted RNA in RNA fragmentation buffer and react at 94℃ for 5 min to break the RNA into small fragments of approximately 150-200nt. Mix the fragmented RNA with the ProtoScript II reverse transcriptase reaction system, which includes reverse transcriptase, primers, and dNTPs (deoxynucleotide triphosphates), to reverse transcribe the fragmented RNA into one-stranded cDNA. Mix the one-stranded cDNA with the NEBNext Second Strand Synthesis two-stranded reverse transcriptase reaction system to synthesize enterovirus double-stranded cDNA. The two-stranded reverse transcriptase reaction system includes two-stranded synthase and dNTPs. Double-stranded cDNA was bound to 1.2 times its volume of magnetic beads. The magnetic beads have specific chemical groups on their surface that can specifically bind to cDNA. Unbound impurities were removed with washing buffer, and finally, the purified cDNA was eluted from the magnetic beads with elution buffer to obtain the purified enterovirus sequence. The purified cDNA was mixed with Qubit reagent, and the fluorescence intensity was measured using a Qubit fluorometer. The concentration of cDNA was calculated according to the standard curve to ensure that the concentration was ≥1 ng / μL.

[0047] (3) Library preparation: Double-stranded cDNA was fragmented using restriction enzymes (Frag / AT Enzymes). The fragmented cDNA was then mixed with end-repair enzymes to concentrate the cDNA fragment length to 200–250 bp, repairing the double-stranded cDNA ends to form blunt ends suitable for adapter ligation. The repaired double-stranded cDNA was mixed with the enzymes, and dATP was added to add an "A" base to the 3' end of the double-stranded cDNA. The cDNA with the "A" tail was then mixed with universal adapters and ligases, allowing the universal adapters to successfully ligate to both ends of the cDNA. The ligation product was mixed with magnetic beads. The magnetic beads have specific chemical groups on their surface that can specifically bind DNA. Unbound impurities were removed with washing buffer, and finally, the purified DNA was eluted from the magnetic beads with elution buffer. The purified cDNA with adapters, obtained after purification with magnetic beads, is the initial library. Using a PCR reaction system with universal adapter primers, the purified cDNA was mixed with the PCR reaction system, including primers, dNTPs, and Taq enzyme, and amplified by PCR to obtain a sufficient initial library concentration of 20 ng / μL.

[0048] (4) Hybridization capture: 500 ng of the initial library (25 μL) was mixed with 4 μL of the enterovirus probe shown in Table 1, and then mixed with the hybridization reaction mixture (Micro Probe Hyb Mixture Plus) for pre-hybridization. Micro Probe Hyb Mixture Plus includes universal blockers, repeat sequence blockers (COT DNA), and adapter blockers (Illumina Adapter Blockers) to obtain the probe-blocker-library complex. The nucleotide sequence of the probe blocker is shown in SEQ ID NO. 31. The hybridization blocking system includes universal blockers (nucleotide sequence shown in SEQ ID NO. 31), repeat sequence blockers (COT DNA, concentration 50 ng / μL), and adapter blockers (concentration 10 μM), which can respectively inhibit non-specific probe binding, repeat sequence interference in the sample, and adapter dimer formation, thereby improving hybridization specificity. MicroProbe Hyb Mixture Plus is a premixed hybridization reaction solution compatible with Illumina / MGI sequencing platforms. Each 490μL tube (16 rxn specification) contains a probe blocker (5μM), a repetitive sequence blocker (COT DNA, 20 ng / μL), an adapter blocker (10 μM), and hybridization buffer (containing Tris-HCl, NaCl, EDTA, etc.). No additional blocking reagents are required; it can be used directly for hybridization reactions. Enterovirus probes are oligonucleotide fragments designed to complement specific RNA or cDNA sequences of target enteroviruses in the initial library. The probes specifically bind to the target sequence, thereby enriching the target sequence from the complex sample background during hybridization. MicroProbe Hyb Mixture Plus is manufactured by Beijing Microbial Technology Co., Ltd. COT DNA is used to block high-copy / repetitive sequences in the human genome, and the adapter blocker is used to block index sequences.

[0049] Probe blockers are oligonucleotide fragments that can bind to non-target sequences in the initial library to prevent probes from binding nonspecifically to non-target sequences, thereby reducing background signals.

[0050] Add 3 times the volume of DNA purification magnetic beads to the pre-hybridized mixture to bind the magnetic beads to the probe-blocker-library complex. Use magnetic bead separation technology to separate the magnetic beads bound to the probe-blocker-library complex from the unbound material to obtain the purified probe-blocker-library complex.

[0051] Add 20 μL of preheated (65℃) Fast Hybridization Mix and 30 μL of Fast Hybridization Enhancer to the system, denature at 95℃, and then hybridize at 60℃ for 2 hours. The Fast Hybridization Mix improves the binding efficiency of the probe to the target sequence, reduces non-specific binding, and ensures that more target sequences are enriched. Fast Hybridization reduces non-specific binding, further improving the specificity of hybridization and reducing background noise.

[0052] After hybridization, 100 μL of streptavidin binding beads and 200 μL of Fast Binding Buffer were added. The streptavidin binding beads were used to target and capture the probe-library complex, thereby enriching the target sequence. The Fast Binding Buffer accelerates the binding reaction, shortens the capture time, and improves experimental efficiency.

[0053] Non-specific bindings were removed by washing at 65 °C (Fast Wash Buffer 1) and at low temperature (Wash Buffer 2), resulting in an enterovirus-enriched library. Fast Wash Buffer 1 is typically used for washing at 65 °C, which effectively removes non-specific binding sequences that bind at lower temperatures. Wash Buffer 2 is typically used for washing at low temperature to further remove any remaining non-specific binding sequences.

[0054] The enriched library was amplified using a high-fidelity enzyme (Equinox Library Amp Mix). After amplification, 90 μL of magnetic beads were added for purification. The purified library was separated from unbound substances using magnetic bead separation technology to obtain a purified enriched library. The purified library was mixed with Qubit reagent, and the fluorescence intensity was measured using a Qubit fluorometer. The concentration of the library was calculated based on the standard curve, and the concentration of the library was found to be 12 nM.

[0055] (5) Sequencing analysis: The library was sequenced on the Illumina NovaSeq platform with sequencing parameters set as follows: read length PE 150, sequencing depth ≥ 500×. The bioinformatics analysis workflow included: raw data filtering (removal of adapter contamination and low-quality reads) → reference genome alignment (using target virus genomes such as poliovirus and Coxsackievirus A16 as references) → genome coverage calculation → variant site detection (using GATK software). A blank control (RNA extraction and library construction without samples) and a negative control (fecal samples from healthy individuals free of enteroviruses) were included to ensure no cross-contamination. The data alignment results are shown in Table 2, with 100% enterovirus genome coverage.

[0056] Table 2 Sequencing data from the Illumina NovaSeq platform Example 2: Preparation of a kit for enterovirus detection 1. Components of the reagent kit (1) RNA pathogen capture library preparation kit (Box1) Table 3 Note: UDI primers are used to add specific sample indexes during library PCR amplification (one pair of UDI primers is used for each sample), which facilitates the splitting of different sample data after high-throughput sequencing; 30 probes are responsible for hybridization to capture target enterovirus sequences. The two functions are independent and work together to complete the library construction process.

[0057] 2. Reagent kit preparation method (1) Preparation of probe mixture: Dissolve 30 core probes in probe preservation solution, adjust the concentration to 10 μM, dispense into 4 μL / tube, and store at -20℃ in the dark; (2) Reagent dispensing and sterilization: Liquid reagents such as lysis buffer and washing buffer are sterilized by filtration through a 0.22μm filter membrane and then dispensed according to specifications; magnetic bead suspension is dispersed by ultrasonication (300 W power, 5 minutes time) and then dispensed; enzyme reagents (endonuclease, PCR enzyme) are dispensed under ice bath conditions and stored at -80℃; (3) Quality control verification: Take one tube of standard and operate according to the kit instructions. The following conditions must be met: DNA concentration after extraction ≥1ng / μL, initial library concentration ≥20 nM, target sequence ratio of enriched library after hybridization capture ≥40%, genome coverage ≥95%, to ensure the kit is qualified; (4) Assembly and packaging: Pack the reagents, instructions and quality control reports of each module into the reagent kit packaging box in order, transport at 4℃ and store at -20℃ for long-term storage (enzyme reagents are stored separately at -80℃).

[0058] Example 3: Virus detection and sensitivity verification 1. Experimental Materials Positive sample: Poliovirus Sabin type 1 positive fecal sample (verified by RT-qPCR, concentration 10). 6 (copies / μL) Negative samples: Stool samples from healthy individuals (verified by RT-qPCR to be free of enteroviruses); This invention includes a kit, a traditional metagenomic sequencing kit, and an RT-qPCR kit.

[0059] 2. Experimental Procedure (1) Sample gradient dilution: Dilute 10 6 Positive samples of copies / μL were serially diluted with enzyme-free water to 10 copies / μL. 5 10 4 Five concentration gradients were set up: 10³, 10², and 10¹ copies / μL, with three replicates for each gradient. A negative control (fecal sample from healthy individuals) and a blank control (enzyme-free water) were also set up.

[0060] (2) Library preparation and sequencing: ① Following the method in Example 1 of this invention, RNA extraction, double-stranded cDNA synthesis, initial library construction, hybridization capture, and enriched library amplification were performed on samples of each gradient dilution, negative control, and blank control to finally obtain the final library; ② Traditional metagenomic sequencing kits were used to construct libraries for the same samples according to their instructions (without hybridization capture steps); ③ All libraries were sequenced on the Illumina NovaSeq platform, with sequencing parameters uniformly set to PE150 and sequencing depth ≥500×.

[0061] (3) Data analysis: ① Filter the raw data to remove low-quality reads and connector contamination; ② Using the Sabin type 1 poliovirus genome (GenBank accession number NC_002058.3) as a reference, the viral sequence percentage, genome coverage, and sequencing depth of each sample were calculated; ③ Judgment criteria: The target virus sequence accounts for ≥0.1% and the genome coverage is ≥80%, which is considered a valid detection.

[0062] 3. Experimental Results Figure 2, the detection limit validation diagram, shows that the method of this invention can still effectively capture viral genomes in samples as low as 10² copies / μL (target sequence percentage ≥ 0.5%, sequencing depth ≥ 50×); traditional metagenomic methods cannot effectively detect viral genomes in samples with a target sequence percentage < 0.1% in samples with a density below 10³ copies / μL. The detection limit of the method of this invention (10² copies / μL) is significantly better than that of traditional metagenomic methods (10³ copies / μL), and the repeatability stability of samples at each gradient is good (RSD < 10%). The negative control and blank control did not detect the target virus, and there was no cross-contamination.

[0063] As can be seen from the examples, the detection method used in this invention has a stronger detection capability for low-load and complex matrix samples compared with traditional methods. It can cover more serotypes, reduce "type false negatives", provide more complete genome coverage, assist in mutation and evolution analysis, be compatible with multiple infection detection, analyze viral population structure, adapt to automation and high throughput, and have high detection efficiency.

[0064] Although the above embodiments have described the present invention and its implementation in detail, it should be noted that for those skilled in the art, any changes, modifications, substitutions, combinations, simplifications, etc., made to the corresponding conditions without departing from the technical principles of the present invention should be considered as equivalent substitutions, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A method for targeted capture and library construction of enteroviruses using liquid-phase probes, characterized in that, Includes the following steps: S1. Sample processing and double-stranded cDNA synthesis: Enterovirus RNA was extracted from the sample using a viral RNA extraction kit, the RNA was fragmented, the fragmented RNA was reverse transcribed into first-stranded cDNA, the first-stranded cDNA was synthesized into double-stranded cDNA of enterovirus and purified to a concentration ≥1 ng / μL. S2. Constructing the initial cDNA library: The double-stranded cDNA is fragmented to a length of 200-250 bp, and the fragments are purified by end repair, A-tailing, adapter ligation, and PCR amplification to obtain the initial library; S3. Probe and library hybridization capture: The initial library is mixed with the probe pool and hybridization blocking system, denatured, and hybridized at 60-65℃ for 1-4 hours. The hybridization product is captured using streptavidin magnetic beads, washed to remove non-specific bindings, and the virus-enriched library is obtained by elution. S4. Post-capture amplification and purification: The enriched library was amplified and purified by PCR to obtain a virus-targeted sequencing library suitable for high-throughput sequencing; The probe pool contains a set of oligonucleotide probes capable of specifically binding to conserved regions or coding regions of enterovirus genomes. The nucleotide sequences of the probes are selected from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 30, or variant sequences having at least 90% sequence identity with them.

2. The method according to claim 1, characterized in that, The enterovirus is one or more of the following: poliovirus, Coxsackievirus A16, echovirus 9, or enterovirus 71.

3. The method according to claim 1, characterized in that, The reverse transcription system described in step S1 includes reverse transcriptase, primers, and deoxyribonucleotide triphosphates; the fragmentation described in step S2 is achieved by enzymatic fragmentation or sonication.

4. The method according to claim 1, characterized in that, The mixing ratio of the initial library and probe pool in step S4 is 500 ng: 4 μL.

5. The method according to claim 1, characterized in that, The hybridization blocking system includes a probe blocker, a repeat sequence blocker, a linker blocker, and a hybridization buffer. The nucleotide sequence of the probe blocker is selected from the sequence shown in SEQ ID NO:

31.

6. A reagent kit for enteroviruses, characterized in that, It includes the probe pool, probe preservation solution, hybridization blocking system, streptavidin magnetic beads, and washing buffer as described in claim 1.

7. The reagent kit according to claim 6, characterized in that, The detection sensitivity for fecal, blood, cerebrospinal fluid, or pharyngeal swab samples can be as low as 10² copies / μL.

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