Capture probe for detecting whole genome of human parainfluenza virus as well as kit and application thereof

Abstract

The invention provides a capture probe for detecting a human parainfluenza virus whole genome and a kit and application thereof, and relates to the technical field of gene sequencing. The capture probe composition disclosed by the invention comprises probes of which the sequences are shown as SEQ ID NO. 1 to SEQ ID NO. 947. The capture probe composition disclosed by the invention can fully cover the full-length genome of the human parainfluenza virus type 1-4, and has the characteristics of uniformity, specificity and high capture efficiency on a captured region. According to the method, the whole genome sequence of the human parainfluenza virus can be more economically and efficiently obtained through probe hybridization capture and next-generation sequencing technologies, and the purposes of human parainfluenza virus variation monitoring and virus type traceability can be achieved depending on sequence information.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene sequencing, and in particular relates to a capture probe for detecting the full-length genome of human parainfluenza virus, a kit and an application thereof. Background Art

[0002] Human parainfluenza viruses (HPIVs) are single-stranded negative-sense RNA viruses belonging to the Paramyxoviridae family. They are the second most common pathogen causing severe acute respiratory diseases in infants and young children worldwide. Currently, four serotypes of HPIVs have been identified, HPIV1-4, of which HPIV4 contains two serosubtypes, HPIV4a and HPIV4b. The genetic differences between different serotypes are large, and the epidemiological characteristics, clinical manifestations, and disease burden of the diseases caused are different. With the widespread prevalence of HPIVs around the world, the existing molecular typing standards can no longer meet the needs of virus tracking and tracing, so there is an urgent need for methods that can perform high-throughput sequencing and analysis of the entire HPIV genome.

[0003] The detection method of probe hybridization capture combined with next-generation sequencing is a high-throughput sequencing technology for analyzing genomic variation and functional elements. In the past few decades, with the development and increasing application of second-generation sequencing technology, people's understanding of variation, mutation and functional elements in genomes and transcriptomes has continued to deepen. However, the detection and identification of large-scale genomic variation and complex functional elements are still challenging. In traditional genome sequencing methods, whole genome sequencing faces high costs and huge data processing problems, while targeted genome sequencing can reduce the amount of sequencing data by selecting gene regions of interest, but it cannot fully cover the entire genome, and the detection of variation and functional elements of the entire genome is limited. Moreover, the method of probe hybridization capture combined with second-generation sequencing still has some challenges in processing virus samples with poor quality or low load. The genomic sequence information of these samples often has insufficient coverage or missing (Gap) in the middle of the genome during the detection process.

[0004] The viral load of clinical samples of HPIVs is low, and it is difficult to obtain virus strain samples due to problems with storage conditions and experimental techniques. As a result, when using traditional nested PCR and first-generation sequencing methods, the primer design is complex, the amplification efficiency is low, the specificity is poor, it is time-consuming and consumable, and it is easy to be contaminated, affecting the accuracy of the results. Ordinary second-generation sequencing also results in insufficient RNA starting amount due to small sample size and low viral load, necessitating an increase in the number of PCR amplification cycles, which in turn generates a large amount of redundant data, affecting the sequencing quality.

[0005] Therefore, the development of capture probes for full-length genome detection of HPIV types 1-4 is of great significance to solving the problems of high cost and excessive data volume of traditional genome sequencing methods and the limitations of targeted sequencing. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a capture probe composition for detecting the whole genome of human parainfluenza virus. The capture probe composition combined with the second-generation sequencing technology can obtain the whole genome sequence of human parainfluenza virus more economically and efficiently.

[0007] Another object of the present invention is to provide an application of a capture probe composition for detecting the whole genome of human parainfluenza virus.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a capture probe composition for detecting the whole genome of human parainfluenza virus. The capture probe composition comprises probes with sequences shown as SEQ ID NO.1 to SEQ ID NO.947.

[0010] Preferably, the capture probe has a biolabel at its 5' end.

[0011] The present invention also provides a kit for detecting the whole genome of human parainfluenza virus, and the kit comprises the capture probe composition.

[0012] Preferably, the kit further comprises streptavidin-labeled magnetic beads and library hybridization reaction reagents.

[0013] The present invention also provides an application of the capture probe composition or the kit in preparing a product for screening human parainfluenza virus.

[0014] The present invention also provides a use of the capture probe composition or the kit in preparing a product for detecting the whole genome of human parainfluenza virus.

[0015] Preferably, the human parainfluenza virus complete genome includes the full-length genome sequences of four serotypes of human parainfluenza virus types 1 to 4.

[0016] The present invention also provides a method for detecting the full-length genome of human parainfluenza viruses 1 to 4 based on capture probes and second-generation sequencing, comprising the following steps:

[0017] The human parainfluenza virus 1-4 genome library of the sample to be tested is hybridized with the capture probe composition to obtain a hybridization reaction solution; the DNA fragments hybridized with the probe are separated from the hybridization reaction solution; after obtaining the DNA fragments hybridized with the probe, the DNA fragments hybridized with the probe are used as templates for post-capture PCR amplification, the obtained PCR products are subjected to second-generation sequencing, and the sequencing results are subjected to data analysis to obtain the full-length genome of the human parainfluenza virus 1-4.

[0018] More preferably, the reaction system for post-capture PCR amplification includes the following reagents in a 50 μL system: 24 μL of DNA fragment suspension hybridized with the probe, 1 μL of Post PCR primers, and 25 μL of Post PCR Master Mix.

[0019] More preferably, the reaction procedure of the post-capture PCR amplification is 95°C for 1 min; 98°C for 20 s, 60°C for 30 s, 72°C for 30 s, 7 to 12 cycles; 72°C for 5 min.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention provides a capture probe composition for detecting the whole genome of human parainfluenza virus. The capture probe composition, combined with the second-generation sequencing technology, can more economically and efficiently obtain the whole genome sequence of human parainfluenza virus, thus solving the detection difficulties caused by the limited sample size and low viral load of human parainfluenza virus (HPIVs).

[0022] (2) The present invention provides a probe hybridization capture system that can cover the full-length genome sequences of four serotypes of human parainfluenza virus types 1-4. Due to the large differences in the genomes of HPIV1-4 viruses, it is not possible to use a set of primers to complete the detection of genome sequences of different serotypes. However, by using the capture probe combination of the present invention in combination with the second-generation sequencing method, a set of probe systems can be used to achieve full coverage of the genome sequences of different serotypes of parainfluenza virus.

[0023] (3) High specificity: The probe hybridization capture system provided by the present invention combined with the second generation sequencing technology can achieve nearly 100% genome coverage for the human parainfluenza virus genome sequence.

[0024] (4) High throughput. The probe hybridization capture system provided by the present invention combined with the second-generation sequencing technology can realize the simultaneous sequencing of multiple samples, which has the advantages of high throughput, simplicity and speed compared with conventional PCR methods. DETAILED DESCRIPTION

[0025] The present invention provides a capture probe composition for detecting the whole genome of human parainfluenza virus. The capture probe composition comprises probes with sequences shown as SEQ ID NO.1 to SEQ ID NO.947.

[0026] In the present invention, the 5' end of the capture probe preferably has a biological marker, more preferably labeled with biotin, and the biotin label facilitates the subsequent separation after the capture probe hybridizes with the target region DNA to form a complex. In the present invention, there are 947 capture probes in total, which can fully cover the HPIV1-4 genome sequence, and have the characteristics of uniformity, specificity and high capture efficiency for the captured area. The capture probe is preferably synthesized by high-throughput electrochemical synthesis. In a specific embodiment of the present invention, the capture probe is commissioned to Aigitaikang Biotechnology (Beijing) Co., Ltd. for synthesis.

[0027] The present invention also provides a kit for detecting the whole genome of human parainfluenza virus, and the kit comprises the capture probe composition.

[0028] In the present invention, the kit preferably further comprises streptavidin-labeled magnetic beads and library hybridization reaction reagents. In the present invention, the streptavidin-labeled magnetic beads bind to the biotin on the capture probe through streptavidin, and then the capture probe and the target region DNA sequence targeted and bound to the capture probe are separated by magnetic field force. The library hybridization reaction reagents preferably include at least one of the following reagents: TargetSeq Hyb Buffer v2, Hyb HumanBlock, Blocking Oligo and RNase enzyme inhibitor. The library hybridization reaction reagent is used for the hybridization reaction of the capture probe, and is used for the capture probe to bind to a specific DNA sequence in the genomic library.

[0029] In the present invention, the kit preferably also includes RNA fragmentation reagents, reverse transcription reaction reagents, cDNA double-strand synthesis reaction reagents, linker connection reagents, purification reagents, PCR pre-reaction reagents and post-capture PCR amplification reaction reagents. The PCR pre-reaction reagents preferably include at least one of the following: PCR Master Mix containing UDG enzyme and UDI primers. The RNA fragmentation reagents, reverse transcription reaction reagents, cDNA double-strand synthesis reaction reagents, linker connection reagents, purification reagents, and PCR pre-reaction reagents are used to construct the HPIV1-4 genomic library. The present invention has no special restrictions on the reagents for constructing the HPIV1-4 genomic library, and reagents well known in the art can be used. The post-capture PCR amplification reaction reagents preferably include at least one of the following: PCR primers and post-capture PCR reaction premix. The post-capture PCR amplification reaction reagents are used to amplify the DNA sequence of the captured target area to obtain a large number of DNA sequences in the target area to meet the sequencing requirements.

[0030] The present invention also provides an application of the capture probe composition or the kit in preparing a product for screening human parainfluenza virus.

[0031] The present invention also provides a use of the capture probe composition or the kit in preparing a product for detecting the whole genome of human parainfluenza virus.

[0032] In the present invention, the human parainfluenza virus complete genome preferably includes the full-length genome sequences of four serotypes of human parainfluenza virus types 1 to 4.

[0033] The present invention also provides a method for detecting the full-length genome of human parainfluenza viruses 1 to 4 based on capture probes and second-generation sequencing, preferably comprising the following steps:

[0034] (1) Hybridizing the human parainfluenza virus 1-4 genomic library of the sample to be tested with the capture probe composition to obtain a hybridization reaction solution; the hybridization reaction system is preferably 30 μL, preferably including the following reagents: TargetSeq Hyb Bufferv213μL, Hyb Human Block 5μL, Blocking Oligo 2μL, RNase Block 5μL, Nuclease-Free Water 3μL and Target Probes 2μL. The hybridization reaction procedure is preferably 80℃5min, 50℃12~18h. There is no special limitation on the method for constructing the human parainfluenza virus 1-4 genomic library of the sample to be tested, and the genomic library construction method known in the art can be used, such as total RNA extraction and fragmentation, reverse transcription and cDNA second-strand synthesis, linker addition and PCR pre-reaction and fragment purification to obtain the genomic library.

[0035] (2) separating the DNA fragments hybridized with the probe from the hybridization reaction solution; the method for separating the DNA fragments hybridized with the probe from the hybridization solution preferably comprises mixing magnetic beads modified with streptavidin with the hybridization solution, combining the magnetic beads with the hybridization complex by utilizing the binding properties of streptavidin and biotin to form a ternary complex, and separating the ternary complex from the hybridization solution under the action of a magnetic field.

[0036] (3) After obtaining the DNA fragment hybridized with the probe, the present invention uses the DNA fragment hybridized with the probe as a template to perform post-capture PCR amplification, and the obtained PCR product is subjected to second-generation sequencing. The sequencing results are subjected to data analysis to obtain the full-length genome of HPIV1-4. In the present invention, the reaction system for post-capture PCR amplification is preferably 50 μL, and preferably includes the following reagents: 24 μL of the DNA fragment suspension hybridized with the probe, 1 μL of PostPCR primers, and 25 μL of Post PCR Master Mix. The reaction procedure for post-capture PCR amplification is preferably 95°C for 1 min; 98°C for 20 s, 60°C for 30 s, 72°C for 30 s, 7 to 12 cycles; 72°C for 5 min. In the present invention, the method for performing data analysis on sequencing results preferably includes preliminary filtering of the original sequencing data to obtain Clean reads; removing the host gene sequence from the Clean reads, aligning the obtained sequence to the viral genome, and assembling to obtain a contig sequence; removing the host gene sequence from the contig sequence again and aligning it to the viral genome sequence to obtain virus typing and sorting data; aligning the virus typing and sorting data to the reference genome, and obtaining the full genome of human parainfluenza virus or the full-length genome of HPIV1-4 after correcting the variant sites.

[0037] In the present invention, since there are adapter information and low-quality sequence reads in the original sequence obtained by sequencing, in order to improve the quality of the sequencing data, the original sequence data is initially filtered. The method of the initial filtering is preferably to cut off the sequence with an average base quality value less than 20bp in an 8bp sliding window manner; remove the adapter sequence at the end of the sequence; if the first base or the last base of the sequence is less than 20bp, the base will be directly cut off; usually, if the remaining sequence length is less than 40 (double-end) after the removal is completed, the pair of sequences will be discarded.

[0038] In the present invention, the method for removing the host gene sequence is preferably to use Bowtie2 software to align the sequencing data to the reference genome of the host, and collect the sequences that cannot be aligned.

[0039] In the present invention, preferably, the collected unaligned sequences are aligned to the viral genome using bwa software. The viral genome refers to the HPIV1-4 genome. The method for assembling contig sequences is preferably assembled using MEGAHIT software.

[0040] In the present invention, the contig sequence is again depleted of the host gene sequence and then aligned to the viral genome to obtain virus typing and sorting data. The viral genome refers to the HPIV1-4 genome.

[0041] In the present invention, clean reads are aligned to the reference genome, and the HPIV1-4 full-length genome is obtained after correction of the variant sites. The alignment is preferably performed using bwa software. The variant site correction is preferably performed using samtools software for mutation analysis, and after obtaining the variant site, iVar software is used to correct the variant site.

[0042] In the present invention, the above-mentioned detection method is adopted to combine probe hybridization capture and second-generation sequencing technology, so that the whole genome sequence of HPIV1-4 can be obtained quickly and at low cost.

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0045] Example 1

[0046] The method of probe hybridization capture combined with second-generation sequencing mainly includes key steps such as probe design, probe preparation, library construction, high-throughput sequencing after PCR amplification, and data analysis. The present invention is based on the human parainfluenza virus type 1-4 downloaded from the NCBI Virus database, including 93 sequences of HPIV1 (Taxonomy ID: 12730), 76 sequences of HPIV2 (Taxonomy ID: 2560525), 447 sequences of HPIV3 (Taxonomy ID: 11216), and 49 sequences of HPIV4 (Taxonomy ID: 2560526). The oligonucleotide probes complementary to the sequences in the database are designed using the principle of base complementary pairing to ensure that all variants are covered. Subsequently, the probes of all variants are merged to remove redundancy to avoid the problem of excessive depth in highly conserved regions. Finally, 947 probes were designed and synthesized, with a coverage of up to 100%, and the capture depth of each region of each genome was uniform and effective.

[0047] During the probe design process, factors such as probe length, number of probe layers, probe specificity, probe binding ability, probe competition resistance, and binding secondary structure need to be considered. In order to achieve multi-level coverage of the target area, the probe sequence shingled design method is adopted, and thermodynamic stability evaluation and probe compensation are performed. In addition, it is necessary to avoid designing probes in low-complexity areas. In special cases, a comprehensive evaluation of the placement position is required. The final designed probes have high enough uniqueness and specificity for different genome ranges.

[0048] 1. Probe design and optimization:

[0049] 1.1 Design probe sequence: Based on the target region information, design a specific probe sequence with a length of 100 nt.

[0050] 1.2 Shingled design: adopts a multi-layer probe coverage strategy to ensure high coverage of the target area.

[0051] 1.3 Thermodynamic evaluation: Evaluate the thermodynamic stability of the probe and design probe compensation for areas of low stability.

[0052] 1.4GC content balance: Based on the characteristics that the region with a GC content of about 50% has a higher binding capacity, the number of probes is appropriately increased to compensate for the competitive resistance and binding capacity of the GC high / low regions.

[0053] 1.5 Specificity: that is, the uniqueness of the probe in the genome. The higher the specificity, the higher the capture efficiency of the probe. Probes should be designed to avoid low-complexity regions; but in special cases, for example, some hot spots happen to be located in low-complexity regions, then it is necessary to comprehensively evaluate the sequence of the region and adjacent regions and place the probes with caution.

[0054] 2. Probe preparation: The template synthesis of the probe adopts a high-throughput electrochemical synthesis method, and the synthesized short sequence oligonucleotides (oligo) are mixed and quality tested. The oligo pool that has passed the quality inspection uses biotin-labeled nucleotide triphosphates (NTPs) and similar nucleic acid substances as synthetic raw materials, and the RNA form of the probe is prepared by in vitro transcription. This method can provide a high-efficiency, high-throughput synthesis process and ensure the consistency and reliability of the probe quality.

[0055] 2.1 Secondary structure: The probe itself should be prevented from forming a hairpin structure and the probes should be prevented from forming dimers, otherwise the binding of the probe to the target DNA will be affected.

[0056] 2.2 For pathogenic microorganisms with multiple highly variable sequences, please note:

[0057] (a) Design probes for all variants to avoid omissions;

[0058] (b) All variant probes are merged to remove redundancy and prevent the depth from being too high in highly conserved regions.

[0059] After comprehensive evaluation, a total of 947 probes were designed and synthesized, with a coverage of up to 100% for all sequences in the database, and ensuring uniform and effective capture depth in each region of the genome. The sequences of the designed capture probes are shown in Table 1.

[0060] Table 1 Nucleotide sequence information of capture probe composition

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[0070]

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[0080]

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[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] Example 2

[0094] A method for detecting the full-length genome of HPIV1-4 based on capture probes and second-generation sequencing, the specific steps are as follows:

[0095] (I) Extraction of genomic RNA from the sample to be tested

[0096] The samples to be tested were clinical samples of HPIV1-4 infected persons collected by the National Laboratory from 2019 to 2023. RNA was extracted from the samples using QIAamp Viral RNA Minikit (Cat. No. 52906, Qiagen, Germany). RNA concentration was quantified using Qubit RNA HS assay kit and Qubit Fluorometer (Life Technologies, USA).

[0097] (II) Library preparation

[0098] 1. RNA fragmentation

[0099] 1.1 Take out the RNA sample from the -80℃ refrigerator in advance, and take out the Fast Frag Buffer from the -20℃ refrigerator in advance, place it on an ice box to melt, vortex briefly to mix and centrifuge it instantaneously, and place it on an ice box for later use.

[0100] 1.2 Prepare the reaction system on an ice box according to Table 2:

[0101] Table 2 Fragmentation reaction system

[0102] Reagents volume RNA samples 13μL (total volume 10ng~1μg) FastFragBuffer 4μL Total volume 17μL

[0103] 1.3 Use a pipette to mix or vortex to mix, and centrifuge briefly.

[0104] 1.4 Set the PCR instrument parameters as follows, place the reaction solution on the PCR instrument, and run the program according to Table 3:

[0105] Table 3 Reaction procedure

[0106]

[0107] 1.5 When the temperature of the PCR instrument drops to 4°C, take out the PCR tube, centrifuge it momentarily, and proceed to STEP 2 immediately.

[0108] 2. Reverse Transcription

[0109] 2.1 Take out the FastFirst Strand Buffer in the kit from the -20℃ refrigerator in advance, place it on an ice box to melt, vortex briefly to mix and centrifuge it instantaneously, and place it on an ice box for later use.

[0110] 2.2 Take out the FastFirst StrandEnzyme in the kit from the -20℃ refrigerator, invert and centrifuge briefly, and place it on an ice box for later use.

[0111] 2.3 Prepare the reaction system on an ice box according to Table 4:

[0112] Table 4 Reverse transcription reaction system

[0113] Reagents volume Sample from the end of STEP1 reaction 17μL FastFirstStrandBuffer 6μL FastFirstStrandEnzyme 2μL Total volume 25μL

[0114] 2.4 Use a pipette to mix (avoid vigorous shaking) and centrifuge briefly.

[0115] 2.5 Set the PCR instrument parameters as follows, place the reaction solution on the PCR instrument, and run the program according to Table 5:

[0116] Table 5 Reverse transcription reaction system

[0117]

[0118] 2.6 When the temperature of the PCR instrument drops to 4°C, take out the PCR tube, centrifuge it momentarily, and proceed to STEP 3 immediately.

[0119] 3. Synthesis of cDNA second strand and addition of "A" at the 3' end

[0120] 3.1 Take out the Fast Second Strand Buffer with dUTP in the kit from the -20℃ refrigerator in advance, place it on an ice box to melt, vortex briefly to mix and centrifuge it instantly, and place it on an ice box for later use.

[0121] 3.2 Take out the Fast Second Strand Enzyme in the kit from the -20℃ refrigerator, invert to mix and centrifuge briefly, and place on an ice box for later use.

[0122] 3.3 Prepare the reaction system on an ice box according to Table 6:

[0123] Table 6 cDNA second-strand synthesis system

[0124] Reagents volume Sample from the end of step 2 reaction 25μL FastSecondStrandBufferwithdUTP 30μL FastSecondStrandEnzyme 5μL Total volume 60μL

[0125] 3.4 Use a pipette to mix (avoid vigorous shaking) and centrifuge briefly.

[0126] 3.5 Set the PCR instrument parameters as follows, place the reaction solution on the PCR instrument, and run the program according to Table 7:

[0127] Table 7 cDNA second-strand synthesis reaction program

[0128]

[0129] 3.6 When the temperature of the PCR instrument drops to 4°C, take out the PCR tube, centrifuge it momentarily, and proceed to STEP 4 immediately.

[0130] 4. Connector connection

[0131] 4.1 Take the Adapter out of the -20℃ refrigerator in advance and place it on an ice box to thaw. After thawing, vortex briefly to mix and centrifuge it instantly, and place it on an ice box for later use.

[0132] 4.2 Dilute the Adapter (15 μM) to an appropriate concentration in advance according to the amount of RNA used for library construction. The dilution multiples are shown in Table 8:

[0133] Table 8 Input RNA amount and adapter ratio

[0134] RNA input amount Connector concentration Dilution multiple 100ng~500ng 7.5μM 2 times 50ng 3.75μM 4 times 25ng 1.5μM 10 times 10ng 0.75μM 20 times

[0135] 4.3 Take out the Fast Ligation Buffer in the kit from the -20℃ refrigerator in advance, place it on an ice box to melt, vortex briefly to mix, centrifuge it instantaneously, and place it on an ice box for later use.

[0136] 4.4 Take out the Fast Ligase Mix from the -20℃ refrigerator, invert to mix, centrifuge briefly, and place on an ice box for later use.

[0137] 4.5 Prepare the reaction system on an ice box according to Table 9:

[0138] Table 9 Linker Addition Reaction System

[0139] Reagents volume Sample from the end of STEP3 reaction 60μL FastLigationBuffer 30μL FastLigaseMix 5μL Adapter (after dilution) 5μL Total volume 100μL

[0140] If a single experiment processes a large number of samples and the reaction solution needs to be pre-mixed, please do not pre-mix the Adapter. The best operation method is to first mix the Adapter (after dilution) with the sample after the Step 3 reaction, and then add the pre-mixed reaction solution, which can effectively reduce the self-ligation of the adapter.

[0141] 4.6 Use a pipette to mix (avoid violent shaking), and centrifuge momentarily.

[0142] 4.7 Set the PCR instrument parameters as follows (close the heated cover or do not cover the heated cover), place the PCR tube on the PCR instrument, and run the program according to Table 10:

[0143] Table 10 Linker addition reaction procedure

[0144]

[0145] 4.8 When the temperature of the PCR instrument drops to 4°C, take out the PCR tube, centrifuge it momentarily, and proceed to STEP 5 immediately.

[0146] 5. Post-ligation purification

[0147] 5.1 Prepare 80% ethanol with anhydrous ethanol and Nuclease-Free Water in advance and keep it at room temperature for later use. Please try to use freshly prepared 80% ethanol for magnetic bead purification. (The magnetic beads used for purification are IGT TM Pure Beads or Agencourt AMPure XP)

[0148] 5.2 Take out the purified magnetic beads from the 4℃ refrigerator in advance, mix them and place them at room temperature for 30 minutes. Vortex the purified magnetic beads that have been equilibrated to room temperature and set aside.

[0149] 5.3 Add 0.45 times volume (45 μL) of purified magnetic beads to 100 μL of reaction solution after STEP 4, mix by pipetting or vortexing, and let stand at room temperature for 5 minutes.

[0150] 5.4 Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to wait for the solution to clarify.

[0151] 5.5 Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 200 μL 80% ethanol solution into the PCR tube, and let it stand for 30 seconds.

[0152] 5.6 Keep the PCR tube on the magnetic rack, discard the supernatant, add 200 μL 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and discard the supernatant.

[0153] 5.7 Cover the tube cap and centrifuge briefly to remove the residual ethanol to the bottom of the tube. Place it on the magnetic rack and carefully use a 10μL pipette to remove the residual ethanol at the bottom, being careful not to absorb the magnetic beads.

[0154] 5.8 Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3 to 5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0155] 5.9 Add 22 μL of Nuclease-Free Water, remove the PCR tube from the magnetic stand, mix by pipetting or vortexing, and let stand at room temperature for 2 minutes.

[0156] 5.10 Centrifuge briefly and place the PCR tube on a magnetic rack for 2 minutes to wait for the solution to clarify.

[0157] 5.11 Use a pipette to aspirate 20 μL of supernatant, transfer it to a new PCR tube, mark it, and prepare for STEP 6.

[0158] 6. Pre-PCR reaction

[0159] 6.1 Take out the PCR Master Mix with UDG in the kit from the -20℃ refrigerator in advance, place it on an ice box to melt, invert and mix after melting, centrifuge it instantly, and place it on an ice box for later use.

[0160] 6.2 Take out the UDIPrimer in the kit from the -20℃ refrigerator in advance, place it on an ice box to melt, vortex briefly to mix, centrifuge it instantaneously, and place it on an ice box for later use.

[0161] 6.3 Prepare the PCR reaction solution on an ice box according to Table 11 below. Please record the Index number used:

[0162] Table 11 Pre-PCR reaction system

[0163] Reagents volume Sample from the end of STEP5 reaction 20μL PCRMasterMixwithUDG 25μL UDIPrimer 5μL Total volume 50μL

[0164] The UDIPrimer sequence is as follows:

[0165] 5'-AATGATACGGCGACCACCGAGATCTACACNACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQ ID NO: 948), in which N represents "i5 Index", and the i5 Index adapter sequence refers to the Index sequence adapter sequence located at the P5 end (close to the P5 primer end of the Flow-cell) during the construction of the Chinese library on the Illumina sequencing platform.

[0166] That is, 5'-AATGATACGGCGACCACCGAGATCTACAC-i5Index-ACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQ ID NO: 948)

[0167] 5'-CAAGCAGAAGACGGCATACGAGATNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 949), in which N represents "i7 Index", and the i7Index adapter sequence is used to distinguish different samples in the Illumina sequencing platform. The i7Index adapter sequence is usually located at the end of the sequencing library and binds to the sequencing primer to ensure that the data of each sample can be correctly distinguished during mixed sequencing.

[0168] That is, 5'-AATGATACGGCGACCACCGAGATCTACAC-i7Index-ACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQ ID NO: 949)

[0169] 6.4 Use a pipette to mix (avoid violent shaking), and centrifuge momentarily.

[0170] 6.5 Set the PCR program as follows, place the PCR tube on the PCR instrument, and run the program according to the records in Table 12 and Table 13:

[0171] Table 12 Pre-PCR reaction program

[0172]

[0173] Table 13 Relationship between sample volume and PCR cycle number

[0174] Input sample size PCR cycle number (library output 1 μg) 500ng 7 250ng 7 100ng 9 50ng 10 25ng 11 10ng 12

[0175] 6.6 When the temperature of the PCR instrument drops to 4°C, take out the PCR tube, centrifuge briefly, and proceed to STEP 7 immediately.

[0176] 7. Purification after PCR amplification

[0177] 7.1 Prepare 80% ethanol with anhydrous ethanol and Nuclease Free Water in advance and keep it at room temperature for later use. Please try to use freshly prepared 80% ethanol for magnetic bead purification. (The magnetic beads used for purification are IGT TM Pure Beads or Agencourt AMPure XP)

[0178] 7.2 Take out the purified magnetic beads from the 4℃ refrigerator in advance, mix them and place them at room temperature for equilibration for 30 minutes. Vortex the purified magnetic beads that have been equilibrated to room temperature and set aside.

[0179] 7.3 Add 0.9 times the volume of purified magnetic beads (45 μL) to the 50 μL reaction solution after Step 6, mix by pipetting or vortexing, and let stand at room temperature for 5 minutes.

[0180] 7.4 Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to wait for the solution to clarify.

[0181] 7.5 Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 200 μL 80% ethanol solution into the PCR tube, and let it stand for 30 seconds.

[0182] 7.6 Keep the PCR tube on the magnetic rack, discard the supernatant, add 200 μL 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and discard the supernatant.

[0183] 7.7 Cover the tube cap and centrifuge briefly to remove the remaining ethanol to the bottom of the tube. Place the PCR tube on the magnetic rack and carefully use a 10μL pipette to remove the remaining ethanol at the bottom, being careful not to absorb the magnetic beads.

[0184] 7.8 Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3 to 5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0185] 7.9 Add 30 μL of Nuclease-Free Water, remove the PCR tube from the magnetic stand, mix by pipetting or vortexing, and let stand at room temperature for 2 minutes.

[0186] 7.10 Centrifuge briefly and place the PCR tube on a magnetic rack for 2 minutes to wait for the solution to clarify.

[0187] 7.11 Use a pipette to aspirate 28 μL of supernatant, transfer it to a new PCR tube and mark it.

[0188] 7.12 Take 1 μL of library and use Qubit ds DNA HS Assay Kit reagent to measure the library concentration on Qubit 4.0 Fluorometer and record the library concentration.

[0189] 7.13 Take 1 μL of sample and use the fragment analyzer to measure the fragment length. After the experiment is completed, you can arrange for sequencing.

[0190] (III) Targeted capture

[0191] 1. Preliminary preparation for hybrid capture experiment

[0192] This experiment requires about two rounds of liquid phase probe hybridization capture experiments.

[0193] 1.1 Take out Hyb Human Block and RNase Block from the -20℃ refrigerator, place them on an ice box to melt, vortex briefly to mix, centrifuge them instantaneously, and place them on an ice box for temporary storage;

[0194] 1.2 Will be equipped with Take the Blocking Oligo out of the -20℃ freezer, place it on an ice box to melt, vortex briefly to mix, centrifuge it instantaneously, and place it on an ice box for temporary storage;

[0195] 1.3 Take out the probe to be used from the -80℃ refrigerator, place it on an ice box to melt, vortex briefly to mix, centrifuge it instantaneously, and place it on an ice box for temporary storage;

[0196] 1.4 Take out the library to be hybridized and captured from the -20℃ refrigerator, place it on an ice box to melt, vortex briefly to mix, centrifuge it instantaneously, and place it on an ice box for temporary storage;

[0197] 1.5 TargetSeq Take out Hyb Bufferv2, melt at room temperature, vortex briefly to mix, and centrifuge briefly. If there is precipitation, transfer TargetSeq Heat Hyb Buffer v2 in a 37°C water bath and use it after the reagent is completely dissolved.

[0198] 2. Library and probe hybridization

[0199] 2.1 When hybridizing a single library, add 750 ng of the library to a PCR tube and mark it; when hybridizing multiple libraries, add 500 ng of each library.

[0200] 2.2 Place the PCR tube into a vacuum concentrator centrifuge, open the PCR tube cover, and concentrate to dryness.

[0201] 2.3 After the library is concentrated, prepare the hybridization reaction solution as shown in Table 14:

[0202] Table 14 Hybridization reaction system

[0203]

[0204]

[0205] 2.4 Add the hybridization reaction solution to the dried library, vortex for 30 seconds to ensure that the DNA dried at the bottom of the tube is dissolved, and centrifuge briefly.

[0206] 2.5 Set the PCR instrument parameters as follows, place the hybridization reaction solution on the PCR instrument, and run the program according to Table 15:

[0207] Table 15 Hybridization reaction program

[0208]

[0209] 2.6 The recommended hybridization time is 12 to 18 hours. Perform STEP 3 30 minutes before the end of the program.

[0210] 3. Preparation before capture experiment

[0211] 3.1 In advance Cap Beads were taken out of the 4°C refrigerator, mixed thoroughly and equilibrated at room temperature for 30 min.

[0212] 3.2 Take out Wash Buffer 1. If there is precipitation, please heat Wash Buffer 1 in a 37℃ water bath and use it after the precipitation is completely dissolved.

[0213] 3.3 TargetSeq Take out Wash Buffer2v2 and preheat it in a 50℃ water bath.

[0214] 3.4 Take 50 μL Add Cap Beads to a new PCR tube and place it on a magnetic rack for 1 min. Wait for the solution to become clear and discard the supernatant.

[0215] 3.5 Remove the PCR tube from the magnetic stand, add 180 μL of Binding Buffer and mix by pipetting or vortexing to resuspend the magnetic beads.

[0216] 3.6 After instant centrifugation, place the PCR tube on a magnetic rack for 1 minute, wait for the solution to become clear, and discard the supernatant;

[0217] 3.7 Repeat steps 3.5 to 3.6 twice, washing the magnetic beads three times with Binding Buffer;

[0218] 3.8 Remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, mix by pipetting or vortexing, and proceed to STEP 4 immediately.

[0219] 4. Target region DNA capture

[0220] 4.1 Keep the hybridization product of STEP 2 on the PCR instrument and add 180 μL of the prepared product of STEP 3 Add Cap Beads to the hybridization product and mix by pipetting.

[0221] 4.2 Cover the tube cap, remove the PCR tube from the PCR instrument, place it on a vertical rotating mixer, rotate at no more than 10 rpm, and combine at room temperature for 30 minutes (if the laboratory does not have a vertical rotating mixer, you can combine at room temperature for 30 minutes, during which time you can mix by turning it upside down several times every 5 minutes).

[0222] 4.3 Remove the PCR tube, centrifuge it instantly, place it on a magnetic rack for 2 minutes, and discard the supernatant after the solution becomes clear.

[0223] 4.4 Remove the PCR tube from the magnetic rack, add 150 μL of Wash Buffer 1 into the PCR tube and gently pipette to mix to resuspend the magnetic beads, replace the tube cap with a new one, and then place it on a vertical rotating mixer to wash at room temperature for 15 minutes, with a speed not exceeding 10 rpm.

[0224] 4.5 Remove the PCR tube, centrifuge it instantly, place it on a magnetic rack for 2 minutes, and discard the supernatant after the solution becomes clear.

[0225] 4.6 Remove the PCR tube from the magnetic stand and add 150 μL of 50°C preheated TargetSeq Wash Buffer 2v2, gently pipette to mix, centrifuge briefly, place on a metal bath, and incubate at 50°C for 10 min.

[0226] 4.7 Remove the PCR tube, centrifuge it instantly, place it on a magnetic rack for 2 minutes, and discard the supernatant after the solution becomes clear.

[0227] 4.8 Repeat steps 4.6 to 4.7 twice, using TargetSeq Wash the magnetic beads three times with WashBuffer 2v250℃.

[0228] 4.9 Keep the PCR tube on the magnetic rack, add 200 μL 80% ethanol into the PCR tube, let it stand for 30 seconds and then completely discard the ethanol solution (a 10 μL pipette can be used to discard the residual ethanol), and dry the magnetic beads at room temperature to allow the residual ethanol to evaporate completely.

[0229] 4.10 Add 24 μL Nuclease-Free Water to the PCR tube, remove the PCR tube from the magnetic stand, vortex briefly to mix and resuspend the magnetic beads, and perform the amplification reaction of STEP 5.

[0230] 4.11 Keep the PCR tube on the magnetic rack, add 200 μL 80% ethanol into the PCR tube, let it stand for 30 seconds and then completely discard the ethanol solution (you can use a 10 μL pipette to discard the residual ethanol), and dry the magnetic beads at room temperature to allow the residual ethanol to completely evaporate (during the drying process, pay attention to the surface of the magnetic beads to avoid over-drying of the magnetic beads).

[0231] 4.12 Add 24 μL Nuclease-Free Water to the PCR tube, remove the PCR tube from the magnetic stand, vortex briefly to resuspend the magnetic beads, and perform the amplification reaction of STEP 5.

[0232] 5. Post-capture PCR Amplification

[0233] 5.1 Take out Post PCR Master Mix and Post PCR Primer from the -20℃ refrigerator in advance, place them on an ice box to melt, and store them on an ice box temporarily after melting.

[0234] 5.2 Before the experiment, please check again whether PostPCR Primer is used correctly, briefly vortex Post PCR MasterMix and PostPCR Primer, and centrifuge instantly.

[0235] 5.3 Prepare the PCR reaction solution according to Table 16. Note that this step is a PCR reaction with magnetic beads:

[0236] Table 16 Post-capture PCR reaction system

[0237] Reagents volume Magnetic bead suspension obtained in STEP4 24μL PostPCR Primer 1μL PostPCRMasterMix 25μL Total volume 50μL

[0238] Among them, the specific sequence of Post PCR Primer is as follows:

[0239] 5'-AATGATACGGCGACCACCGA (SEQ ID NO: 950)

[0240] 5'-CAAGCAGAAGACGGCATACGA (SEQ ID NO: 951)

[0241] 5.4 After preparation, use a pipette to mix, and quickly transfer to the PCR instrument. Do not use the vortex followed by centrifugation method to mix.

[0242] 5.5 Set the PCR instrument program as follows, place the PCR reaction solution on the PCR instrument, and run the program according to Table 17:

[0243] Table 17 PCR reaction program

[0244]

[0245] After the 5.6 procedure is completed, proceed to the magnetic bead purification in STEP 6.

[0246] The number of PCR cycles can refer to the probe TargetProber tube wall label parameters or probe description files. The number of Post-PCR cycles is related to the total amount of library input during hybridization. When the amount of library input during hybridization is large, the number of Post-PCR cycles can be appropriately reduced. Because the amount of library required for sequencing on the MGI platform is large, it is recommended to add two more cycles.

[0247] 6. Post-amplification purification

[0248] 6.1 Take out the purified magnetic beads, mix well and equilibrate at room temperature for 30 minutes.

[0249] 6.2 Add 1.1 times the volume of magnetic beads (55 μL) to the PCR product of STEP 5, pipette or vortex to mix, and let stand at room temperature for 5 minutes.

[0250] 6.3 Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to wait for the solution to become clear.

[0251] 6.4 Keep the PCR tube on the magnetic rack, discard the supernatant, add 200 μL 80% ethanol solution into the PCR tube, and let it stand for 30 seconds.

[0252] 6.5 Keep the PCR tube on the magnetic rack, discard the supernatant, add 200μL 80% ethanol solution to the PCR tube again, let it stand for 30s and then completely discard the supernatant (you can centrifuge it instantly, centrifuge the hanging liquid to the bottom of the tube, and then use a 10μL pipette to discard the ethanol solution remaining at the bottom).

[0253] 6.6 Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3 to 5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely (during the drying process, pay attention to the surface of the magnetic beads to avoid over-drying of the magnetic beads).

[0254] 6.7 Add 25 μL of Nuclease-Free Water, remove the PCR tube from the magnetic stand, pipette or vortex to mix, and let stand at room temperature for 2 minutes.

[0255] 6.8 Centrifuge briefly and place the PCR tube on a magnetic rack for 2 minutes to wait for the solution to clarify.

[0256] 6.9 Use a pipette to aspirate 23 μL of supernatant and transfer it to a new PCR tube; store the captured library in a -20°C refrigerator. The captured library can be stored in a -20°C refrigerator for one month.

[0257] 6.10 Take 1 μL of library and use Qubit dsDNA HS Assay Kit reagent to measure the library concentration on Qubit 4.0 Fluorometer and record the library concentration.

[0258] 6.11 Take 1 μL of library and use fragment analyzer to check the fragment quality. The fragment size should be basically consistent with the pre-library size.

[0259] On-line sequencing.

[0260] 1 μL was taken from the probe hybridization capture library and quantified using the Qubit ds DNA HS Assay Kit, and the library concentration was recorded; 1 μL of the sample was taken and the fragment length was measured using the Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit), and the library length was between 250 bp and 400 bp. The probe hybridization capture library was sequenced using the NovaSeq 6000 (Illumina) sequencing platform to obtain the raw sequencing data.

[0261] (IV) Data Analysis

[0262] In the raw data obtained by sequencing, there will be a small number of reads containing adapter information, low-quality bases, etc. In order to ensure the quality of information analysis, it is necessary to perform preliminary filtering on the Raw reads to obtain Clean reads, and subsequent analysis is based on Clean reads. The content of data filtering is mainly as follows: using an 8bp sliding window, cut off the sequences with an average base quality value less than 20; remove the adapter sequence at the end of the sequence; if the first base or the last base of the sequence is less than 20, the base will be directly cut off; usually, if the remaining sequence length is less than 40 (double-end) after the removal is completed, the pair of sequences will be discarded.

[0263] Removing host sequences: Since the host genome sequences will still remain in the sequencing data, Bowtie2 sequencing data is first used to align to the host reference genome to obtain the unaligned sequences (unmapR1 & unmapR2). This part of the unmap sequence is the viral sequence information that we may actually need.

[0264] Virus genome alignment: Use bwa software to align the unmapped sequence to the viral genome (the reference species sequence information is designed as the alignment template), and MEGAHIT software to assemble the reads aligned to the genome into contig sequences. Then use blastn to align the contig sequences with the host genome, remove the host sequences again, and obtain the non-human contig file. Then align the non-human contig sequence to the viral genome sequence to obtain the possible virus typing and sorting results-virus.txt.

[0265] Obtain the full-length genome sequence of HPIV1-4: use the results-virus-top1 typing sequence ranked first in the possible virus typing ranking as the reference genome, use bwa software to align the clean reads to the reference genome to obtain the aligned bam file, use samtools software to perform mutation analysis to obtain the variant sites, use iVar software to correct the variant sites of the genome to obtain the final HPIV1-4 full-length genome sequence. The results are shown in Table 18.

[0266] Table 18 Full-length genome sequencing results of HPIV1-4 samples to be tested

[0267]

[0268]

[0269] Comparative Example 1

[0270] A method for detecting HPIV1-4 genomes based on first-generation sequencing comprises the following steps:

[0271] Since HPIVs virus isolation is difficult, clinical samples are often used directly for subsequent testing. However, due to its low viral load and high CT value, the laboratory often uses the nested PCR method to perform two rounds of amplification of the target gene fragments. Among the structural proteins encoded by the HPIVs genome, the HN and F genes show greater antigenicity and genetic variation in the HPIVs genome. Therefore, the control sample uses the nested RT-PCR method to amplify the nucleotide sequences of the HN and F genes of HPIVs, and then the amplified products are sequenced using the first-generation sequencing technology. After splicing the sequencing results, the gene fragments at both ends with greater genetic variation in the HPIVs genome are obtained and compared with the results of the second-generation sequencing.

[0272] 1. Sample description: Sample 1 is HPIV1, sample 2 is HPIV2, sample 3 is HPIV3, and sample 4 is HPIV4.

[0273] 2. Kit: PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa, Dalian, China)

[0274] The specific sequences of the primers for HPIV1-4 nested PCR amplification of the HN gene are shown in Tables 19-22. In the sequences shown in Tables 19-22, Y, R, D, K, M and W are all degenerate bases, the base corresponding to Y is C or T, the base corresponding to R is A or G, the base corresponding to D is G or A or T, the base corresponding to K is G or T, the base corresponding to M is A or C, and the base corresponding to W is A or T.

[0275] Table 19 Primer sequences for nested PCR amplification of the HN gene of HPIV1

[0276]

[0277] Table 20 Primer sequences for nested PCR amplification of the HN gene of HPIV2

[0278]

[0279] Table 21 Primer sequences for nested PCR amplification of the HN gene of HPIV2

[0280]

[0281] Table 22 Primer sequences for nested PCR amplification of the HN gene of HPIV2

[0282]

[0283] The specific sequences of primers for nested PCR amplification of the F gene by HPIV1-4 are shown in Tables 23-26.

[0284] Table 23 Primer sequences for nested PCR amplification of the F gene of HPIV1

[0285]

[0286] Table 24 Primer sequences for nested PCR amplification of the F gene of HPIV2

[0287]

[0288] Table 25 Primer sequences for nested PCR amplification of the F gene of HPIV3

[0289]

[0290]

[0291] Table 26 Primer sequences for nested PCR amplification of the F gene of HPIV4

[0292]

[0293] 3. Use the PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa, Dalian, China) from TaKaRa. Prepare the PCR amplification reaction system according to Table 27, and perform nested PCR amplification according to the reaction program in Table 28.

[0294] Table 27 PCR amplification reaction system

[0295] Reagents Volume (μl) Buffer 12.5 Taq 1 HPIVsF (10 μM) (HPIVsN-F2nd) 1 HPIVsR (10 μM) (HPIVsN-R2nd) 1 RNA 2 <![CDATA[RNaseFreedH2O]]> 7.5 Total 25

[0296] Table 28 HPIVs nested PCR amplification reaction program

[0297]

[0298] 4. Sequence determination: The obtained PCR product was sent to Biotechnology Co., Ltd. for sequence determination.

[0299] 5. Sequence splicing and organization: Sequencher5.0 software was used to organize and splice the sequences. To ensure accuracy, each fragment was tested in both forward and reverse directions.

[0300] 6. Sequence the obtained sample fragments and splice them to obtain the first-generation whole genome sequence, which is compared with the whole genome sequence obtained based on probe hybridization capture and second-generation sequencing technology to verify the sequence consistency.

[0301] Among them, the total length of the target gene HN and F gene fragments in sample 1 is 3396bp, and there are no different sites in the genome sequences obtained by the two sequencing methods, with an accuracy of 100%; the total length of the target gene HN and F gene fragments in sample 2 is 3372bp, and there are no different sites in the genome sequences obtained by the two sequencing methods, with an accuracy of 100%; the total length of the target gene HN and F gene fragments in sample 3 is 3343bp, and there are no different sites in the genome sequences obtained by the two sequencing methods, with an accuracy of 100%; the total length of the target gene HN and F gene fragments in sample 4 is 3355bp, and there are no different sites in the genome sequences obtained by the two sequencing methods, with an accuracy of 100%.

[0302] By comparing the experimental results of Example 1 and Comparative Example 1, it is shown that the present invention can achieve the purpose of accurately and efficiently detecting the HPIV1-4 genome based on probe hybridization capture and second-generation sequencing technology.

[0303] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A capture probe composition for detecting the whole genome of human parainfluenza virus, characterized in that: The capture probe composition includes probes with sequences shown as SEQ ID NO.1 to SEQ ID NO.

947.

2. The capture probe composition according to claim 1, characterized in that The capture probe has a biolabel at its 5' end.

3. A kit for detecting the whole genome of human parainfluenza virus, characterized in that: The kit comprises the capture probe composition according to any one of claims 1 to 2.

4. The kit according to claim 3, characterized in that The kit also includes streptavidin-labeled magnetic beads and library hybridization reaction reagents.

5. Use of the capture probe composition according to any one of claims 1 to 2, or the kit according to any one of claims 3 to 4 in the preparation of a product for screening human parainfluenza virus.

6. Use of the capture probe composition according to any one of claims 1 to 2, or the kit according to any one of claims 3 to 4, in the preparation of a product for detecting the whole genome of human parainfluenza virus.

7. The use according to claim 6, characterized in that: The human parainfluenza virus full genome includes the full-length genome sequences of four serotypes of human parainfluenza virus types 1 to 4.

8. A method for detecting the full-length genome of human parainfluenza viruses 1 to 4 based on capture probes and second-generation sequencing, comprising the following steps: The human parainfluenza virus 1-4 genome library of the sample to be tested is hybridized with the capture probe composition according to any one of claims 1 to 2 to obtain a hybridization reaction solution; the DNA fragments hybridized with the probes are separated from the hybridization reaction solution; after obtaining the DNA fragments hybridized with the probes, the DNA fragments hybridized with the probes are used as templates for post-capture PCR amplification, the obtained PCR products are subjected to second-generation sequencing, and the sequencing results are subjected to data analysis to obtain the full-length genomes of human parainfluenza viruses 1-4.

9. The method according to claim 8, characterized in that The reaction system of post-capture PCR amplification includes the following reagents in a 50 μL system: 24 μL of DNA fragment suspension hybridized with the probe, 1 μL of Post PCR primers and 25 μL of Post PCR Master Mix.

10. The method according to claim 8, characterized in that The reaction procedure of the post-capture PCR amplification is 95°C for 1 min; 98°C for 20 s, 60°C for 30 s, 72°C for 30 s, 7 to 12 cycles; 72°C for 5 min.

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