Adenovirus liquid-phase probe targeted capturing and library building method and kit for adenovirus liquid-phase probe targeted capturing and library building method

Abstract

The invention discloses a liquid-phase probe targeted capturing and library building method for adenovirus. The method comprises the following steps: S1, probe design: S1, designing a plurality of biotin-labeled adenovirus probes; s2, sample treatment and DNA purification; s3, carrying out fragmentation treatment on the double-chain cDNA, and carrying out terminal repair and linker connection, PCR amplification and magnetic bead purification to obtain an initial library; s4, mixing the initial library with an adenovirus probe, a blocker and a blocking mixed solution, hybridizing, purifying by magnetic beads, adding a fast hybrid mixed solution and a fast hybrid enzyme to promote hybridization, finally adding streptavidin binding beads and a fast capture buffer solution, and eluting to obtain an adenovirus enriched library; and S5, amplifying the enriched library by using a high-fidelity enzyme, and adding magnetic beads for purification. According to the present invention, with the liquid phase probe targeting capture technology, the core pain points such as low-load missing detection, incomplete serotype coverage, genome information loss, poor high-throughput adaptability and the like in the adenovirus detection are effectively solved, and the core technical support is provided for the adenovirus molecular diagnosis and the public health prevention and control.

Description

Technical Field

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

[0002] Adenovirus belongs to the genus Adenovirus of mammals in the family Adenoviridae. It is a non-enveloped double-stranded DNA virus with a genome length of about 30-38kb. Currently, 57 serotypes have been identified, which are divided into 7 groups (AG) based on differences in gene sequence. It has broad host specificity and pathogenicity, and poses a significant threat to children, the elderly, and immunocompromised individuals.

[0003] 1. Common pathogenic serotypes and groupings Group A: mainly includes types 12, 18, and 31, which mostly cause gastrointestinal infections in infants and young children, and some types are associated with malignant tumors such as bladder cancer in children.

[0004] Group B: encompasses types 3, 7, 11, 14, and 35, and consists of the main pathogens causing respiratory and urinary tract infections. Types 7 and 14 can cause severe pneumonia with a high mortality rate.

[0005] Group C includes types 1, 2, 5, and 6, which mostly cause upper respiratory tract infections in children and can remain dormant in the body for a long time. They are prone to recurrence when immune function declines.

[0006] Group D: Represented by types 8, 19, and 37, it is the primary cause of epidemic keratoconjunctivitis, highly contagious, and prone to outbreaks in crowded places.

[0007] Group F: Types 40 and 41 are the core, second only to rotavirus, and is the second leading cause of viral gastroenteritis in infants and young children. It manifests as watery diarrhea and vomiting, and the course of the disease can last for 1-2 weeks.

[0008] Groups E and G: The clinical detection rate is low. Type 4 in Group E is occasionally seen in respiratory infections, while type 52 in Group G is associated with severe infections in immunocompromised individuals.

[0009] The clinical manifestations of adenovirus infection vary significantly depending on serotype, site of infection, and host immune status. Common diseases include: Respiratory diseases: accounting for more than 60% of adenovirus infections, mainly manifested as the common cold, acute pharyngitis, tonsillitis, and bronchitis. Severe cases can develop into viral pneumonia, with symptoms such as high fever, difficulty breathing, and lung rales. The mortality rate for infants and young children and those with immunodeficiency can reach 10%-30%.

[0010] Gastrointestinal diseases: Infant gastroenteritis caused by F group 40 / 41 is characterized by watery diarrhea, vomiting, and low-grade fever, which can easily lead to dehydration; A group 12 / 31 can cause acute gastroenteritis in adults.

[0011] Eye diseases: Epidemic keratoconjunctivitis caused by type 8 / 37 in group D is characterized by red, swollen, and painful eyes with increased discharge. In severe cases, it can lead to corneal damage and leave visual impairment. Pharyngoconjunctival fever caused by type 3 / 7 in group B is often accompanied by fever and sore throat and is more common in children.

[0012] Urinary system diseases: Hemorrhagic cystitis caused by group B 11 / 21, manifested as gross hematuria, urinary frequency, and dysuria, has a high incidence in children and the course of the disease is about 1-2 weeks.

[0013] Other diseases: Immunocompromised individuals are prone to systemic infections that can affect the liver, spleen, and central nervous system, leading to severe conditions such as liver failure and encephalitis, with extremely high mortality rates.

[0014] Currently, the mainstream detection methods for human adenovirus include cell culture, serological detection, and real-time quantitative PCR (qPCR). The advantages and limitations of each technology are as follows: (1) Cell culture is the gold standard for detecting adenovirus. Its core advantage is that it can obtain live pathogens, which can be further used for serotype identification and drug sensitivity testing, providing a direct basis for the formulation of clinical drug resistance treatment plans.

[0015] This technology has significant shortcomings: First, the experimental cycle is extremely long, requiring 5-14 days for respiratory samples and 14-21 days for fecal samples due to their complex matrix, which completely fails to meet the rapid diagnostic needs of clinical emergencies and cluster outbreak control. Second, it has stringent requirements for culture conditions, requiring the use of specialized adherent cells and demanding high levels of laboratory biosafety and operator skill, making it difficult for grassroots laboratories to implement. Third, the inhibitors in the samples have strong interference; polysaccharides in feces, mucoproteins in eye swabs, and inflammatory cell debris in respiratory samples easily inhibit viral proliferation, resulting in low sensitivity and the ability to detect only 10% of viruses. 4 Samples with a viral load of more than 10 copies / mL are insufficient to meet the criteria for latent infection (viral load <10). 3 The need for testing copies / mL and early infection samples.

[0016] (2) The advantage of serological testing is that it is simple to operate, requiring only basic equipment such as an enzyme-linked immunosorbent assay (ELISA) reader, without expensive instruments, and with low reagent costs. It is suitable for retrospective epidemiological surveys and can be used to trace and analyze the adenovirus infection rate and prevalent serotypes in a specific area (such as a school or community). However, the limitations of this technology are also quite prominent: First, it cannot distinguish between recent and past infections, because antibodies can persist in the human body for 6-12 months after adenovirus infection. If antibodies are detected positive, it is impossible to determine whether it is a recent infection or past immunity, which can easily lead to misjudgment of the infection time. Second, the cross-reactivity rate is high. There is antigenic cross-reactivity with Chlamydia trachomatis, Chlamydia pneumoniae, and other adenovirus serotypes, with a false positive rate as high as 15%-20%, which affects the accuracy of the test results.

[0017] (3) Real-time quantitative PCR (qPCR) is currently the most widely used technology in clinical practice. Its advantages are reflected in three aspects: First, the detection speed is fast, and it only takes 2-4 hours from sample processing to result output, which can quickly provide reference for clinical diagnosis and epidemic control; Second, the sensitivity is relatively high, and the detection limit can reach 10. 3 copies / mL, superior to cell culture method (10 copies / mL). 4 (copies / mL) and serological detection methods (10 copies / mL) 5 The technology has several advantages: firstly, it can cover most overtly infected samples; secondly, it has high specificity, allowing primers to be designed targeting conserved adenovirus genes (such as Hexon, Penton, and 16S rRNA genes) to effectively distinguish adenovirus from other pathogens such as influenza virus, respiratory syncytial virus, and enterovirus, reducing misdiagnosis. However, this technology also has limitations: firstly, primers rely on conserved sequences, and if the adenovirus undergoes gene mutations, the primers may not bind effectively, leading to false negatives, especially for novel recombinant strains with a high rate of missed detection; secondly, complex samples can cause significant interference, such as polysaccharides in feces and mucus proteins in respiratory samples, which can inhibit Taq enzyme activity, resulting in decreased PCR amplification efficiency and a false negative rate of 20%-30%; and thirdly, the genomic information coverage is limited, only able to detect single or a few gene fragments, and cannot obtain the complete adenovirus genome sequence, which is not conducive to subsequent serotyping, transmission chain tracing, and drug resistance mutation detection. Summary of the Invention

[0018] To address the shortcomings of the aforementioned technologies, this invention proposes an integrated workflow of "specific probe design - sample processing - library preparation - hybridization capture - amplification and purification," which can achieve highly sensitive detection of adenoviruses, broad-spectrum serotype coverage, and whole-genome analysis.

[0019] To achieve the objectives of this invention, a method for targeted capture and library preparation of adenovirus using liquid-phase probes is proposed, comprising the following steps: S1. Probe design: Based on conserved regions of the genomes of various types of adenoviruses and the ORF coverage regions of the whole genome, multiple biotin-labeled adenovirus probes were designed; S2. Sample processing and DNA purification: DNA was extracted from the adenovirus sample and purified using magnetic beads to obtain the purified DNA sequence; S3. Library preparation: The DNA sequences obtained above are fragmented to a length of 200-250 bp, and end repair, A-tailing and adapter ligation are performed. After PCR amplification and purification with magnetic beads, the initial library is obtained. S4. Hybridization and capture: The initial library was mixed with adenovirus probes, blocking agents and blocking mixtures in a certain proportion. After hybridization, the mixture was purified by magnetic beads, and then rapid hybridization mixture and rapid hybridization enzyme were added. After denaturation at 95°C, hybridization was carried out at 60°C for 2 hours. Finally, streptavidin binding beads and rapid capture buffer were added. Non-specific bindings were removed by high-temperature washing and low-temperature washing. After elution, the adenovirus enriched library was obtained. S5. Post-capture amplification and purification: The enriched library was amplified using a high-fidelity enzyme, purified by adding magnetic beads, and then subjected to concentration determination and sequencing analysis. The nucleotide sequences of the adenovirus probe are shown in SEQ ID NO.1 to SEQ ID NO.31.

[0020] The probe is 100 bp in length, with a Tm value of 60-65℃ and a GC content of 45%-55%. It has no self-complementary sequences or cross-probe homologous sequences. All probe sequences have <80% homology with the whole genome sequences of closely related pathogens in the NCBI nr database, with a hybridization signal percentage of ≤0.1% and a cross-reactivity rate of less than 0.1%. Closely related pathogens include influenza virus, respiratory syncytial virus, and enterovirus. The probe is biotinylated and specifically binds to streptavidin magnetic beads.

[0021] In step S3, the concentration of double-stranded cDNA is not less than 1 ng / μL.

[0022] In step S3, the double-stranded cDNA is fragmented using an endonuclease, and an "A" tail is added after the ends are repaired.

[0023] In step S4, the initial library and probe are mixed at a ratio of 500 ng: 4 μL, and the hybridization time is 2 hours.

[0024] The blocking mechanisms include: repetitive sequence blocking mechanisms, universal blocking mechanisms, and adapter blocking mechanisms. The host genome repetitive sequences targeted by the universal blocking mechanisms include human Alu sequences and gut microbiota repetitive sequences. The repetitive sequence blocking mechanisms include calf thymus DNA fragments. The adapter blocking mechanisms are completely complementary to the universal adapter sequences used in library construction.

[0025] This invention also proposes an adenovirus liquid-phase probe targeted capture sequencing kit, comprising, (1) Adenovirus liquid-phase probe library; (2) Streptavidin magnetic beads; (3) Hybridization buffer, blocking agent and blocking mixture, washing buffer; (4) PCR Master Mix and magnetic bead purification reagents for enriched library amplification; (5) Instructions for use, which specify the above-described method for preparing a targeted capture library for adenovirus using liquid-phase probes, in order to obtain a targeted capture library that can be directly sequenced.

[0026] Furthermore, the blocking agents and blocking mixtures include universal blockers, repetitive sequence blockers (COT DNA), and adapter blockers; the washing buffers include FastWash Buffer 1, Wash Buffer 2, Stringent Wash Buffer, and TE buffer.

[0027] Furthermore, the kit is used for the enrichment, sequencing, and typing of adenoviruses in feces, sputum, or bronchoalveolar lavage fluid.

[0028] The kit can detect no less than 10 2 Adenovirus copies / μL.

[0029] The beneficial effects of this invention are as follows: This invention solves the problems of low viral load missed detection, incomplete serotype coverage, missing genomic information, and difficulty in high-throughput adaptation in adenovirus detection through the "liquid phase probe targeted capture" technology. Compared with traditional detection technologies, the advantages of this invention in sensitivity, specificity, information richness, and high-throughput adaptation make it a core technical means for adenovirus molecular diagnosis and public health prevention and control. Attached Figure Description

[0030] Figure 1 This is a design diagram for the probe.

[0031] Figure 2 Document library construction flowchart. Detailed Implementation

[0032] To make the objectives, technical solutions, and technical effects of the embodiments of this application clearer, the technical solutions in the embodiments of this application are described clearly and completely. The embodiments described below are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art in conjunction with the embodiments of this application without creative effort are within the scope of protection of this application. Unless otherwise specified, the reagents and equipment used in this invention are conventional reagents and equipment in this technical field. Unless otherwise specified, the strains, reagents, etc. used in this invention are all commercially available.

[0033] Example 1: Adenovirus probe capture and library construction 1. Probe Design Based on conserved regions and whole genome sequences of 57 serotypes in adenovirus group A and B (reference genome accession numbers: Group A serotypes 12 NC_001460.1, 18 NC_001761.1, 31 NC_001802.1; Group B serotypes 3 NC_001467.1, 7 NC_001469.1, 11 NC_001471.1, 14 NC_001436.1, 35 NC_001901.1; Group C serotypes 1 NC_002041.1, 2 serotypes...). Probes were designed using the following genes: NC_001405.1, NC_001406.1 (type 5), NC_005820.1 (type 6); NC_005136.1 (type 8), NC_001491.1 (type 19), NC_001526.1 (type 37) (type 40), NC_001454.1 (type 41), NC_001288.1 (type 4) (type 4) (type 4) (type 4) (type 4) (type 4) (type 5) (type 52), NC_003266.1 (type 5). The specific design parameters are as follows: (1) Length: 100bp, to avoid non-specific binding caused by being too short (<60bp, fewer hydrogen bonds, easy to dissociate) or too long (>150bp, easy to form stem-ring secondary structure); (2) Tm value: 60-65℃ (matching the hybridization temperature), calculated by the Nearest-Neighbor method; (3) GC content: 45%-55% (to avoid the formation of secondary structures), excluding extreme regions in the genome with GC content <30% or >60%; (4) No self-complementary sequences or cross-probe homologous sequences (homology < 80%). (5) Coverage density: Design one probe every 50 bp, with a 50 bp overlap between probes, to ensure that each ORF region is covered by at least 2 probes; (6) Cross-reactivity control: All candidate probe sequences were compared with the whole genome sequences of closely related pathogens (influenza virus, respiratory syncytial virus, enterovirus, etc.) in the NCBI nr database using BLASTN to screen probes with homology <80%; in the in vitro validation stage, the probes were hybridized with pure culture DNA of closely related pathogens, and probes with a hybridization signal ratio >0.1% were removed to ensure that the cross-reactivity rate was less than 0.1%.

[0034] Thirty-one biotin-labeled DNA probes were designed, and their nucleotide sequences are shown in the table below: Table 1 Probe nucleotide sequences 2. Sample preparation Take 0.2g of fecal sample from an infant with gastroenteritis, add 2mL of physiological saline, vortex for 10 minutes, centrifuge at 12000rpm for 10 minutes at 4℃, and collect 200μL of supernatant. Use a magnetic bead DNA extraction kit to extract total DNA from the elution buffer containing viral DNA, obtaining 30μL of elution buffer. Add twice the volume of magnetic beads to the extracted total DNA for binding. The magnetic beads have specific chemical groups on their surface, which can specifically bind to double-stranded cDNA virus. Use washing buffer to remove unbound impurities, and finally use elution buffer to elute the purified cDNA from the magnetic beads to obtain the purified adenovirus sequence. Mix the purified cDNA with Qubit reagent, measure the fluorescence intensity using a Qubit fluorometer, and calculate the concentration of double-stranded cDNA according to the standard curve, ensuring that the concentration is ≥1 ng / μL.

[0035] 3. Library preparation Double-stranded cDNA was fragmented using restriction enzymes (Frag / AT Enzymes), with fragment lengths concentrated between 200–250 bp. End-repair enzymes were used to fill in the 3′ concave ends and remove the 3′ convex ends, resulting in blunt ends for all fragments. Polynucleotide kinases were used to phosphorylate 5′-OH to 5′-P, providing substrate for subsequent adapter ligation. dNTP Mix was used as a polymerase substrate to further blunt the ends, and an “A” tail was added to the 3′ end of the blunt-end fragments to form a 3′-A overhang. Universal adapters were ligated to both ends of the above fragments. The sequences were then purified using magnetic beads to remove free adapters, enzymes, and salts. Washing with 80% ethanol removed PEG, ATP, and proteins, keeping the DNA on the magnetic beads. Elution followed by low-salt desorption yielded an initial library with adapters. PCR amplification of the pre-library was performed, followed by magnetic bead purification to obtain the initial library (concentration: 20 ng / μL).

[0036] 4. Hybrid capture Take 1 μL of 500 ng initial library and mix it with 4 μL of biotin-labeled adenovirus whole genome probe, 5 μL of universal blockers, 1 μL of repeat sequence blockers (COT DNA) and 1 μL of adapter blockers, for a total volume of 12 μL, to obtain the probe-blocker-library complex.

[0037] Adenovirus probes: complementary to the specific cDNA sequence of the target adenovirus in the initial library, specifically binding to the target sequence to achieve enrichment; Universal blocking agent (concentration 5μM): targets repetitive sequences in the host genome (human Alu sequences, gut microbiota repetitive sequences) that are commonly found in the sample, preventing the probe from binding to the host nucleic acid; Repeat sequence blocker (concentration 100ng / μL): calf thymus DNA fragment, rich in repeat sequences, can bind to repeat sequence binding sites that may exist in the probe, reducing probe self-aggregation; Adapter blocker (concentration 10 μM): Fully complementary to the universal adapter sequence used in library construction, preventing nonspecific binding of the probe to the library adapter sequence.

[0038] 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.

[0039] 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. 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.

[0040] Non-specific bindings were removed by washing at 65 °C (Fast Wash Buffer 1) and at low temperature (Wash Buffer 2), and the adenovirus-enriched library was obtained after elution. Fast Wash Buffer 1 is usually used for washing at 65 °C, which can effectively remove non-specific binding sequences that bind at lower temperatures. Wash Buffer 2 is usually used for washing at low temperature to further remove any remaining non-specific binding sequences.

[0041] 5. Post-capture amplification and purification The enriched library was amplified using a high-fidelity enzyme (Equinox Library Amp Mix). The amplified library was pre-denatured at 95°C for 3 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 8 cycles. After amplification, 90 μL of magnetic beads were added for purification. Magnetic bead separation was used to separate the purified library from unbound substances, yielding a purified enriched library. The purified library was then mixed with Qubit reagent, and the fluorescence intensity was measured using a Qubit fluorometer. The library concentration was calculated based on a standard curve, yielding a concentration of 12 nM.

[0042] 6. Sequencing and Analysis (1) Sequencing parameters: The library was sequenced in pairs (2×150bp) on the Illumina NovaSeq platform, with a single sample sequencing data volume of 1.5Gb and a sequencing quality requirement of Q30≥85%; (2) Bioinformatics analysis process: Raw data filtering: Trimmomatic software was used to remove low-quality reads (Q<20), adapter sequences, and contaminated sequences; Target sequence enrichment: The filtered data is compared with the probe sequences (SEQ ID NO.1~31) to capture homologous sequences (comparison threshold identity≥95%). Genome assembly: Genome assembly was performed using SPAdes 3.15.5 software to obtain scaffolds sequences; Gene annotation: Structural genes and virulence-related genes were annotated using the Adenovirus Database, and whole-genome functional annotation was performed using Prokka software; Serotyping: Based on the SNP sites of the Hexon and Penton genes, the serotype is determined by comparison with the adenovirus serotype database (AdV Serotype Database); (3) Reference settings: Blank control: Nuclease-free water was used to replace the sample, and the entire experimental procedure was followed to verify that the reagents and operations were free of contamination. Negative control: Fecal samples known to be free of adenovirus (verified negative by cell culture and qPCR) were used to verify capture specificity and avoid false positives; (4) Detection results: Adenovirus type 41 (F group) genome coverage reached 98.2%, target sequence accounted for 42.3%, and viral load was 8.6×10 3The coverage of Hexon, Penton, and 16S rRNA genes all reached 100%, and the genotype was successfully classified as group F41, which perfectly matched the clinical symptoms (watery diarrhea) and had no host nucleic acid interference.

[0043] Example 2: Preparation of a kit for adenovirus detection 1. Components of the reagent kit 1.1 RNA Pathogen Capture Library Preparation Kit (Box 1) Table 2 Note: UDI primers are index primers for library PCR amplification, used to distinguish different samples (adapted to multiplex sequencing). Their sequences have no homology with the Campylobacter jejuni genome and are only responsible for introducing sequencing adapters and sample tags. The 31 probes are capture probes targeting the adenovirus genome, responsible for specifically binding to the target sequence. The two are functionally independent and work together to complete the "library construction-capture" process.

[0044] 2. Reagent kit preparation method 2.1 Preparation of probe mixture: 31 biotin-labeled adenovirus DNA probes (100 bp in length) were mixed at equimolar concentrations, added to probe preservation solution, and the final probe concentration was adjusted to 10 ng / μL. After aliquoting, the mixture was stored at -20℃. 2.2 Buffer preparation: Rapid capture buffer, high-temperature washing buffer, low-temperature washing buffer, washing buffer, and elution buffer were prepared separately, filtered through a 0.22 μm filter membrane for sterilization, and stored at 4°C. 2.3 Enzyme and magnetic bead preparation: High-fidelity enzymes, endonucleases, and rapid enzyme catalysts were mixed with enzyme preservation solution and the final concentration was adjusted to 1 U / μL. The mixture was then stored at -20℃. The magnetic beads were resuspended in washing buffer and the concentration was adjusted to 10 mg / mL. The mixture was then stored at 4℃. 2.4 Kit Assembly: Dispense the probe mixture, probe preservation solution, various buffer solutions, enzymes, magnetic beads, universal adapters, and Qubit reagents according to the specified proportions, seal the package with the accompanying instruction manual, and store at -20℃ in the dark.

[0045] Example 3: Sensitivity Verification Experiment (1) Sample gradient dilution: Adenovirus type 7 (Group B, known concentration 10) was diluted with adenovirus type 7 (Group B, known concentration 10). 6 (copies / μL) serially diluted with enzyme-free water 10 6 10 5 10 4 10 3 10 2 10 1copies / μL; (2) Library construction and detection: The method of this invention was used to construct libraries for samples of various dilutions, with three replicates for each concentration; the method of this invention was used to construct libraries for samples of various concentrations and to sequence them, while traditional qPCR (target gene Hexon) and metagenomic sequencing were used as controls; the results of the detection of samples of various concentrations are shown in Table 3: Table 3. Quantitative Table of Detection Results for Samples of Different Concentrations (3) Results analysis: 10 2 The capture success rate for samples with concentrations of copies / μL and above was 100%, with a genome coverage of ≥95%; 10 1 The sample capture success rate of 33% (copies / μL) demonstrates that the detection limit of this invention is 10. 2 The sensitivity is significantly better than traditional qPCR (10-fold) and metagenomic sequencing (100-fold) than that of traditional qPCR.

[0046] Example 4: Serological Coverage Validation (1) Sample selection: Positive samples of 20 serotypes of adenovirus AG group (types 1, 3, 7, 8, 11, 12, 14, 18, 21, 31, 35, 37, 40, 41, 52, 55, 57 and 3 novel recombinant strains) were selected. (2) Library construction and sequencing: The method of this invention was used to construct the library and perform sequencing to analyze the capture efficiency and coverage of each serotype; (3) Results analysis: The capture efficiency of all 20 serotypes was ≥85%, the genome coverage was ≥92%, and the coverage of the novel recombinant strain reached 96.5%, proving that the present invention can achieve broad-spectrum coverage of all adenovirus serotypes.

[0047] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for targeted capture and library preparation of adenovirus using liquid-phase probes, characterized in that, Includes the following steps: S1. Probe design: Based on conserved regions of the genomes of various types of adenoviruses and the ORF coverage regions of the whole genome, multiple biotin-labeled adenovirus probes were designed; S2. Sample processing and DNA purification: DNA was extracted from the adenovirus sample and purified using magnetic beads to obtain the purified DNA sequence; S3. Library preparation: The DNA sequences obtained above are fragmented to a length of 200-250 bp, and end repair, A-tailing and adapter ligation are performed. After PCR amplification and purification with magnetic beads, the initial library is obtained. S4. Hybridization and capture: The initial library was mixed with adenovirus probes, blocking agents and blocking mixtures in a certain proportion. After hybridization, the mixture was purified by magnetic beads, and then rapid hybridization mixture and rapid hybridization enzyme were added. After denaturation at 95°C, hybridization was carried out at 60°C for 2 hours. Finally, streptavidin binding beads and rapid capture buffer were added. Non-specific bindings were removed by high-temperature washing and low-temperature washing. After elution, the adenovirus enriched library was obtained. S5. Post-capture amplification and purification: The enriched library was amplified using a high-fidelity enzyme, purified by adding magnetic beads, and then subjected to concentration determination and sequencing analysis. The nucleotide sequences of the adenovirus probe are shown in SEQ ID NO.1 to SEQ ID NO.

31.

2. The method according to claim 1, characterized in that, The probe is 100 bp in length, with a Tm value of 60-65℃ and a GC content of 45%-55%. It has no self-complementary sequences or cross-probe homologous sequences. All probe sequences have <80% homology with the whole genome sequences of closely related pathogens in the NCBI nr database, with a hybridization signal percentage of ≤0.1% and a cross-reactivity rate of less than 0.1%. Closely related pathogens include influenza virus, respiratory syncytial virus, and enterovirus. The probe is biotinylated and specifically binds to streptavidin magnetic beads.

3. The method according to claim 1, characterized in that, In step S3, the concentration of double-stranded cDNA is not less than 1 ng / μL.

4. The method according to claim 1, characterized in that, In step S3, the double-stranded cDNA is fragmented using an endonuclease, and an "A" tail is added after the ends are repaired.

5. The method according to claim 1, characterized in that, The blocking mechanisms include: repetitive sequence blocking mechanisms, universal blocking mechanisms, and adapter blocking mechanisms. The host genome repetitive sequences targeted by the universal blocking mechanisms include human Alu sequences and gut microbiota repetitive sequences. The repetitive sequence blocking mechanisms include calf thymus DNA fragments. The adapter blocking mechanisms are completely complementary to the universal adapter sequences used in library construction.

6. A kit for adenovirus liquid-phase probe-targeted capture library construction, characterized in that, Includes the adenovirus probe and probe preservation solution as described in any one of claims 1-4, as well as hybridization buffer, blocking agent, blocking agent mixture, washing buffer, etc.

7. The reagent kit according to claim 6, characterized in that, Detectable of no less than 10 2 Adenovirus copies / μL.

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