CN118957165A - Probe combination, kit and detection method for detecting influenza A virus - Google Patents

Abstract

The invention provides a probe combination, a kit and a detection method for detecting influenza A virus. Wherein the probe combination comprises a probe for capturing the HA gene and a probe for capturing the NA gene, and the probe combination comprises a probe sequence shown as SEQ ID NOs:1-198, and a combination of nucleic acid sequences as set forth in seq id no. Can solve the problem that the prior art is difficult to quickly capture all known subtypes of the influenza A virus, and is suitable for the field of gene sequencing.

Description

Probe combination, kit and detection method for detecting influenza A virus

Technical Field

The invention relates to the field of gene sequencing, in particular to a probe combination, a kit and a detection method for detecting influenza A virus.

Background

Influenza (Influenza), an acute respiratory infectious disease caused by Influenza virus. Influenza viruses are classified into three types of A type (A type), B type (B type) and C type (C type) according to the difference of surface proteins; the influenza A virus and the influenza B virus are main viruses which cause seasonal influenza epidemics, the variation of the influenza B virus can generate a new main stream strain, but cross immunity exists between the new strain and the old strain, namely the immune reaction aiming at the old strain is still effective on the new strain; influenza A virus is a type with most frequent mutation, antigen drift occurs in the subtype, usually once in 2-3 years, usually causes seasonal or local epidemic, and generates a large antigenicity mutation every ten years, thus generating a new strain.

Influenza a virus contains 8 negative strand RNA genome segments, three medium-sized RNA segments encoding HA, NA and NP, with hemagglutinin (Hemagglutinin, HA) and Neuraminidase (NA) being key surface proteins of the influenza a virus. Hemagglutinin is columnar and is a trimer, and covers the whole surface of the virus at basically the same interval, HA is combined with host surface protein in the process of virus infection, and mediates the combination of a virus membrane and a host cell nuclear membrane, and plays an important role in the process of virus infection; neuraminidase is a tetramer consisting of four subunits, which is not evenly distributed on the viral surface, but rather is polymerized into groups, and participates in viral replication in a multiplexed manner, facilitating release of viral particles from infected host cells. Influenza a viruses are classified into different subtypes according to the difference in hemagglutinin and neuraminidase on the surface of the virus. Serotypes are divided according to HA and NA genes, where HA (1-16) includes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, NA (1-11) includes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, N11.

Currently, the major pandemic in the population is influenza a virus (Influenza A virus) of some subtypes H1N1 and H3N2, and so on. Currently, HA and NA genetic variation tracking for influenza a viruses is mainly viral cell culture, serological diagnostic techniques, sanger sequencing, and targeted NGS detection techniques. The virus cell culture is a classical method, the virus is separated from infected tissue cells for identification, although the result is reliable, the time is long, the cell culture generally needs 3-4 days, and the separation culture of the high avian influenza virus needs to be carried out in a biosafety tertiary laboratory (BSL-3), so that the method is not suitable for rapid detection of the virus. Serological diagnostic techniques include hemagglutination and inhibition assays (HI), agar diffusion Assays (AGP), ELISA methods, neutralization assays (NT), immunofluorescence techniques (IFA), etc., which are well-specific and are commonly used to identify subtypes, but serological methods generally require longer incubation and processing. Sanger sequencing protocol was as follows: the specific primer is used for amplifying NA and NA genes, the carrier is constructed, sanger sequencing is carried out, the technical process is complicated, virus subtypes are required to be identified according to serotypes, and then the specific primer is used for amplifying, the primer is required to be designed aiming at a conserved sequence, and the phenomenon that the amplification of pathogenic genome cannot be completed due to virus variation often occurs. Targeting NGS sequencing is classified into multiplex PCR amplification and liquid phase hybridization capture techniques, wherein multiplex PCR amplification requires coverage of primers of all subtypes, aiming at the phenomenon that mutant strains are missed or cannot complete amplification of full-length genomes; the liquid phase hybrid capture technology designs a specific probe according to a target sequence, captures the target for sequencing through specific combination of the probe and the target, has certain fault tolerance, and can track the virus variation, but the traditional liquid phase hybrid capture technology has a complicated flow and limits the application of the traditional liquid phase hybrid capture technology in the pathogen detection field.

In summary, the methods in the prior art are difficult to rapidly and accurately identify all known subtypes of influenza a virus, and development of a new detection method is urgently needed.

Disclosure of Invention

The invention mainly aims to provide a probe combination, a kit and a detection method for detecting influenza A virus, which are used for solving the problem that in the prior art, all known subtypes of the influenza A virus are difficult to rapidly capture.

In order to achieve the above object, according to a first aspect of the present invention, there is provided a probe set for detecting influenza a virus, the probe set comprising a probe for capturing HA gene and a probe for capturing NA gene, the probe set comprising a probe set as shown in SEQ ID NOs:1-198, and a combination of nucleic acid sequences as set forth in seq id no.

Further, the probes in the probe combination each comprise a biomarker.

Further, the biomarker comprises biotin.

In order to achieve the above object, according to a second aspect of the present invention, there is provided a kit for detecting influenza a virus, the kit comprising the above probe combination for detecting influenza a virus.

Further, the kit also comprises any one or more of the following components: hybridization reaction solution, elution buffer, streptavidin magnetic beads, nucleic acid purification magnetic beads, reverse transcription buffer, PCR buffer, reverse transcriptase, DNA polymerase, end repair enzyme, end repair reaction buffer, molecular tag-containing linker, library amplification primer, linker blocker, DNA blocker, hybridization enhancer or capture library PCR primer.

In order to achieve the above object, according to a third aspect of the present invention, there is provided a method for detecting influenza a virus, the method comprising: a) Extracting RNA in a sample and carrying out reverse transcription to obtain a cDNA library; b) Mixing and hybridizing the probe combination with the cDNA library to obtain a probe-target fragment complex; the probe combination is the probe combination for detecting the influenza A virus or the probe combination in the kit for detecting the influenza A virus; c) Performing PCR amplification on the probe-target fragment complex to obtain a capture library; d) Sequencing the capture library to detect influenza A virus in the sample.

Further, influenza a virus consists of HA subtypes and NA subtypes, the HA subtypes being selected from any one or more of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16; the NA subtype is selected from any one or more of N1, N2, N3, N4, N5, N6, N7, N8 or N9.

Further, influenza a viruses include any one or more of H1N1, H3N2, H3N8, H5N1, H9N2, or H7N 9.

Further, the detection method is a detection method for non-diagnostic or therapeutic purposes.

Further, b) comprises: combining and mixing the cDNA library with a probe to obtain a hybridization system; placing the hybridization system at 55-65 deg.C for hybridization for 0.5-2 hr; after the hybridization reaction is finished, capturing and purifying the probe-target fragment complex in the hybridization system by using magnetic beads to obtain the probe-target fragment complex.

By using the technical scheme of the invention, the probe combination can realize the specific capture of all influenza A virus subtypes, further realize the typing identification of the influenza A virus in a single sample, has high detection speed and can meet the detection timeliness of epidemiological pathogens.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

fig. 1 shows a schematic diagram of a detection method according to an embodiment of the invention.

Fig. 2 shows a diagram of the detected frequency result according to embodiment 1 of the present invention.

Figure 3A shows an influenza genome coverage and depth of coverage results graph according to example 2 of the present invention.

Figure 3B shows an influenza genome coverage and depth of coverage results graph according to example 2 of the present invention.

Figure 3C shows an influenza genome coverage and depth of coverage results graph according to example 2 of the present invention.

Figure 3D shows an influenza genome coverage and depth of coverage results graph according to example 2 of the present invention.

FIG. 3E shows a graph of variant site population results according to example 2 of the present invention.

Fig. 4 shows the sample detection results of the multiple mixed infection influenza strain according to example 3 of the present invention.

Fig. 5 shows the depth of coverage results of the detection method according to example 4 of the present invention on influenza a virus.

Fig. 6A shows the depth of coverage results for influenza a virus according to the detection method of example 4 of the present invention.

Fig. 6B shows the depth of coverage results for influenza a virus according to the detection method of example 4 of the present invention.

Fig. 6C shows the depth of coverage results for influenza a virus according to the detection method of example 4 of the present invention.

Detailed Description

It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to examples.

As mentioned in the background, it is difficult in the prior art to accomplish the identification of the various subtypes in a short time for influenza viruses, especially influenza a viruses. In order to overcome the above problems, the present inventors have attempted to develop a probe combination for detecting influenza a virus, and on the basis of this, developed a liquid phase hybridization capture technique with high detection sensitivity and simple flow for genotyping identification of influenza a virus. Thus, a series of protection schemes of the present application are presented.

In a first exemplary embodiment of the present application, there is provided a probe set for detecting influenza a virus, the probe set comprising an HA gene probe and an NA gene probe, the probe set comprising the nucleic acid sequences as set forth in SEQ ID NOs:1-198, and a combination of nucleic acid sequences as set forth in seq id no.

The specific sequences of the probes are shown below.

SEQ ID NO:1:tgaactattactggacactagtagaaccgggagac,SEQ ID NO:2:aataacattcgaagcaactggtaatctagtggcac,SEQ ID NO:3:aggtatgcattcacaatggaaaaagaagctggatct,SEQ ID NO:4:tattatcatttcagatacaccagtccacaattgcaatgca,SEQ ID NO:5:ttgtcagacacccgagggtgctataaacaccagcc,SEQ ID NO:6:ccatttcaaaatgtacatccgatcacgattgggaaat,SEQ ID NO:7:ccaaagtatgtaagaagcacaaaattgagactggc,SEQ ID NO:8:cccagagtcaatgggcaaagagggagaatggaatt,SEQ ID NO:9:cttggactctattagatacatgggatgtcataaactttga,SEQ ID NO:10:gcactggtaatttaattgcaccagaatatggattcaaaat,SEQ ID NO:11:caaagagaggaagctcaggaattatgaagacagag,SEQ ID NO:12:aacacttgaaaattgtgagaccagatgtcagaccc,SEQ ID NO:13:ttaggggcaataaatacaacattgcccttccacaa,SEQ ID NO:14:ttcacccactgacaataggtgagtgccccaaatat,SEQ ID NO:15:tacgaagtgggaaaagctcaataatgagatcagatg,SEQ ID NO:16:cccattggcaaatgtaagtctgaatgcatcactcc,SEQ ID NO:17:atggaagcatttccaatgacaaaccgttccaaaatgtaaa,SEQ ID NO:18:ggatcacatacggggcctgtcccaaatatgttaag,SEQ ID NO:19:aagcaccctgaaactggcaacaggaatgcgaaatg,SEQ ID NO:20:ccagagaaacaaaccagaggcatatttggcgcaat,SEQ ID NO:21:cgggtttcatagaaaatggatgggagggaatggtg,SEQ ID NO:22:tggttggtacggtttcaggcatcaaaattctgagg,SEQ ID NO:23:gactgattcggagatgaacaagctctttgaaagag,SEQ ID NO:24:aggcgacaactcagggagaatgctgaggacaaagg,SEQ ID NO:25:atgggtgctttgaaatattccacaagtgtgacaacaa,SEQ ID NO:26:gcattgaaagcattcggaatgggacctatgatcat,SEQ ID NO:27:tgtttatagagatgaagcgatcaacaaccgattccaaata,SEQ ID NO:28:gggagtcaaattgacccaggggtacaaggacatca,SEQ ID NO:29:ctttggatttcgttctccatatcatgctttttgctc,SEQ ID NO:30:gcccaagacatactggaaaaagcacacaacgggaa,SEQ ID NO:31:tctgtgatctaaatggggtgaagcctctgattttaaa,SEQ ID NO:32:attgtagtgtagctggatggctcctcggaaaccca,SEQ ID NO:33:gtgcgacgaattcatcagagtgccggaatggtcct,SEQ ID NO:34:atagtggagcgggctaatccagctaatgacctctg,SEQ ID NO:35:acccagggagcctcaatgactacgaagaactgaaa,SEQ ID NO:36:cctgttgagcagaataaatcactttgagaagattctgat,SEQ ID NO:37:tccccaagagttcctggccaaatcatgaaacatca,SEQ ID NO:38:agaacctgcatgaaagggtcaaatcacaactgaga,SEQ ID NO:39:caatgctaatgacttagggaacgggtgctttgaat,SEQ ID NO:40:tggcataagtgtgacaatgaatgtatggagtcagt,SEQ ID NO:41:aaaatggtacctttaactaccccaagtatcaagcc,SEQ ID NO:42:gagtaggttaaacagacagaaaatagaatcagtaaaattg,SEQ ID NO:43:ggagtttggcgtgtatcaaatccttgccatatata,SEQ ID NO:44:agggtatggagcagtctagttgtggtagggctgat,SEQ ID NO:45:cggcaatgggtctttggatgtgttcaaatggctca,SEQ ID NO:46:attcgctctgattacgaccattccgacaaatgcag,SEQ ID NO:47:aaaatctgcctcggacatcactccgtgtcaaacgg,SEQ ID NO:48:ccaaagtaaacacattaactgaaaaaggagtggaagtc,SEQ ID NO:49:caatgcaaccgaaacagtggaacgaacaaacaccc,SEQ ID NO:50:aggatctgctcaaaagggaaaaagacagttgacct,SEQ ID NO:51:gtcaatgtggactcctgggaacaatcactggacca,SEQ ID NO:52:tcaatgtgaccaattcctaaaattttcagccgatttaatt,SEQ ID NO:53:acatgattctaatgtcaaaaacctgtttgatgaagtaagg,SEQ ID NO:54:gagactttctgccaatgcaatagatgctgggaatg,SEQ ID NO:55:tgcttcgatatacttcacaaatgtgacaatgaatgcatg,SEQ ID NO:56:gactataaagaatgggacttatgatcataaggagtatgag,SEQ ID NO:57:agaggccaaactggaaaggagcaagatcaatggag,SEQ ID NO:58:aaactagaggaaaataccacctataagattctcagcattt,SEQ ID NO:59:agtacagtggcggccagtctttgcttggcaatcct,SEQ ID NO:60:agagcgttgggttccaatgctgtggaagatggaaa,SEQ ID NO:61:ggtgtttcgagctatatcacaaatgtgatgaccaat,SEQ ID NO:62:atggagacaattcgaaatgggacctataacaaaaggaaat,SEQ ID NO:63:caagaggagtcaaaattggaaagacagaaaatagaagg,SEQ ID NO:64:tcaagttggaatctgaaggaacttacaaaatcctcac,SEQ ID NO:65:tttattcgactgtcgcctcatctcttgtgattgca,SEQ ID NO:66:ggggtttgctgcctttttgttctgggccatgtcca,SEQ ID NO:67:atcaagctgagctattggtagcaatggagaaccag,SEQ ID NO:68:cacaatcgatatggctgattcagaaatgttgaatctatat,SEQ ID NO:69:aagagtgagaaaacaactcaggcaaaatgcagaagaa,SEQ ID NO:70:tgggaaaggatgtttcgaaatatatcacacttgtgatgat,SEQ ID NO:71:atgcatggagagcataagaaacaacacatacgacc,SEQ ID NO:72:tcacagtacagagaagaagctattttaaaaagactgaata,SEQ ID NO:73:aacccagtgacactctcttctggctataaagacat,SEQ ID NO:74:gaggatgctaaaggacaatgccaaagatgagggaa,SEQ ID NO:75:ggatgtttcaccttttaccataagtgtgacaacaaatg,SEQ ID NO:76:tcgaaagggttaggaatgggacatacgatcacaaa,SEQ ID NO:77:attcgaggaagaatctaaaatcaatcgtcaggagatc,SEQ ID NO:78:gggagtgaaattagactccaatgggaatgtatataaaata,SEQ ID NO:79:gtcaatttacagctgcattgcaagcagtctcgtat,SEQ ID NO:80:gcagcactcatcatgggtttcatcttctgggcgtg,SEQ ID NO:81:cggatcagaagggtgttaaaagaaaacgcaatagata,SEQ ID NO:82:ggagatgggtgttttgaaatactacataaatgcaatgatg,SEQ ID NO:83:tgcatgaatacaatcaaaaacgggacttataaccacca,SEQ ID NO:84:actacgaagaagagagtaggcttgagcgacaaaga,SEQ ID NO:85:taatggagtgaaacttgaagagaactctacatataaaatc,SEQ ID NO:86:gagcatctacagcagtgttgcctcaagcttagtat,SEQ ID NO:87:ctgctcatgattattgggggtttcattttcggatg,SEQ ID NO:88:gtacttaagcacaaactcatcagaaagggttgaca,SEQ ID NO:89:ctgttagagaataatgtcccggttacaagctctgt,SEQ ID NO:90:acttggttgagactaaccacacaggaacatattgtt,SEQ ID NO:91:ttgggtggaattagtccggtgcatttgggagactg,SEQ ID NO:92:gcttcgagggctggattctagggaaccctgcctgt,SEQ ID NO:93:cagcaacctggggattagagaatggtcatatttga,SEQ ID NO:94:gaagacccttctgctcctcatggattgtgctatcc,SEQ ID NO:95:gagagttagacaacaatggagaattgaggcacttattta,SEQ ID NO:96:caaagttgcaacaggaagagtaacggtgtctaccc,SEQ ID NO:97:tcagatcaaatcagcattattcccaacataggaagtaga,SEQ ID NO:98:aagagtgaggaaccagagcggcagaataagcatct,SEQ ID NO:99:tggactctagtaaacccaggggattccatcatctt,SEQ ID NO:100:acagcatcgggaacttaatcgcaccaagaggccac,SEQ ID NO:101:caaaataagcaaatccacaaggagcactgtgctta,SEQ ID NO:102:agtgacaagaagattggatcatgcacaagcccttg,SEQ ID NO:103:taaccgataaaggttcaatccaaagtgataaaccttt,SEQ ID NO:104:aggatcgacttccattggatgctactagacccagg,SEQ ID NO:105:atacagtcacttttaccttcaatggtgcgttcatag,SEQ ID NO:106:cctgatagagccagcttcctccgctctaatgccct,SEQ ID NO:107:caggagttgaatacaatgggaaatcactgggaata,SEQ ID NO:108:gagtgatgcacaactcgatgactcatgtgaagggg,SEQ ID NO:109:tgtttctacagtggaggaacgattaatagcccgtt,SEQ ID NO:110:cgttccaaaacatcgatagcagagcagttggaaaa,SEQ ID NO:111:tcccagatatgtgaagcagtcaagtctcccactgg,SEQ ID NO:112:cagaaatctacatgatcaggtcaaaagggcattgaa,SEQ ID NO:113:ataatgcaattgacgaaggagatggttgcttcaatctt,SEQ ID NO:114:acacaaatgcaatgactcatgtatggaaaccattagaaat,SEQ ID NO:115:tacctacaatcatgaagattaccaggaagaatcacaact,SEQ ID NO:116:agagacaggaaattgggggaataaaattaaagactgaaga,SEQ ID NO:117:atgtttataaaatactatcgatttatagctgcattgcaag,SEQ ID NO:118:gtattgtgctggtaggtctcatacttgcatttataatgt,SEQ ID NO:119:gcaagtgcttgtcatgatggcaccaattggctaac,SEQ ID NO:120:ttggaatttctggcccagacagtggggcagtggct,SEQ ID NO:121:gttaaaatacaatggcataataacagacactatcaagagt,SEQ ID NO:122:gaggaacaagatattgagaacacaagagtctgaatgt,SEQ ID NO:123:atgtgtaaatggttcttgctttaccataatgaccgatg,SEQ ID NO:124:ccaagtgatggacaggcctcatacaaaatcttcag,SEQ ID NO:125:tagagaagggaaagataatcaaatcagtcgaaatgaagg,SEQ ID NO:126:gtgttcctttccatctggggaccaagcaagtgtgc,SEQ ID NO:127:agcatggtccagctcaagttgtcacgatggaaaag,SEQ ID NO:128:tggctgcatgtttgtataacgggggatgataaaaat,SEQ ID NO:129:aactgctagcttcatttacaatgggaggcttgtag,SEQ ID NO:130:agtgttgtttcatggtccaacgatattctcagaac,SEQ ID NO:131:aggagtcagaatgcgtttgtatcaatggaacttgt,SEQ ID NO:132:agtagtaatgactgatggaaatgctacaggaaaagct,SEQ ID NO:133:tactaaaatactattcattgaggaggggaaaatcgttc,SEQ ID NO:134:cgatgggaaggaatggatgcatatttgtatgactg,SEQ ID NO:135:aacgacaatgatgcgagtggccaaataatatatgca,SEQ ID NO:136:gagaatgacagactccattagatcatggaaaaaggatata,SEQ ID NO:137:aagaactcaagagtctgaatgtcaatgcattgacg,SEQ ID NO:138:acctgtgttgtagctgtaacagatggtcctgcggc,SEQ ID NO:139:gtagtgcagaccaccgaatctactggatacgagaa,SEQ ID NO:140:taaggtaatgaagtatgaaaacattcccaaaacaaagata,SEQ ID NO:141:ggttcgaaatggtctgggatgcaaatggatgggtg,SEQ ID NO:142:aacggataaggattcaaatggtgtgcaggacattata,SEQ ID NO:143:caatgacaattggtctggttatagtgggagtttca,SEQ ID NO:144:attagaggagaaacaactggtaggaactgcactgt,SEQ ID NO:145:catgtttttgggttgagatgataagggggcaacct,SEQ ID NO:146:ggaaaaaactatatggactagcggtagtagtattgcattt,SEQ ID NO:147:tggtgttaattctgataccacaggttggtcgtggc,SEQ ID NO:148:ggttcaagagggcatgtctttgttataagagagcc,SEQ ID NO:149:ttgtggcttgtggtccttcagagtgcaggacattt,SEQ ID NO:150:cttaactcaaggcgctctgttgaatgataagcatt,SEQ ID NO:151:aacaatacggtaaaagacaggagtccctatcgagc,SEQ ID NO:152:taatgagcgtgccattgggatcctctcccaatgct,SEQ ID NO:153:ccaagccaaatttgagtccgtcggatggtctgcta,SEQ ID NO:154:gcctgccatgacgggaaggagtggatggctattgg,SEQ ID NO:155:tgagtggtgcagacaatgatgcatatgccgtcatc,SEQ ID NO:156:catatcaaggatgtcaatatgcatctcaggaccga,SEQ ID NO:157:aacaatgcatcagcagtggtgtggtacgggggaag,SEQ ID NO:158:cagtaacagaaatcccatcatgggcagggaacatt,SEQ ID NO:159:taggactcaagaatcagaatgtgtatgccataacg,SEQ ID NO:160:gtctgtccagtggtcatgacagatggtcctgcaaa,SEQ ID NO:161:atagggcagaaactaaaataatttattttaaagagggaaa,SEQ ID NO:162:tacagaaaatcgaggaattgaagggtaacgctcaa,SEQ ID NO:163:catcgaggaatgttcgtgttatggatcaggaggga,SEQ ID NO:164:cccagatcaagaagtggatttgaaatgttgaaaataccta,SEQ ID NO:165:gcaggtactgatcccaattctagaatagcagaacg,SEQ ID NO:166:aagaaattgtcgacaacaataattggtcaggctattcc,SEQ ID NO:167:aagcttcattgactattgggacgataacagtgaatg,SEQ ID NO:168:acaacccgtgtttctacgtagagttaattagaggaa,SEQ ID NO:169:cccgaagaggctaaatatgtgtggtggacaagtaa,SEQ ID NO:170:gtttaattgccctatgtgggagcccattcccagtt,SEQ ID NO:171:gaccattagcagaacgtcaagatcgggttttgaaa,SEQ ID NO:172:ttgaaagtcagaaatggctgggtacaaaacagtaaaga,SEQ ID NO:173:aaatcaaaaggcaagttgtggtcgataactcaaattggt,SEQ ID NO:174:ggatacagtggttctttcacactaccagtggagtt,SEQ ID NO:175:aaaaaacgatatggacctcaagtagctccattgtgat,SEQ ID NO:176:gtggagtagactatgagattgccgactggtcatgg,SEQ ID NO:177:acatcctcgaggtctgggtacgagatgttaaaagt,SEQ ID NO:178:caaatgcattgacagatgatagatcaaagcccatc,SEQ ID NO:179:aggtcagacaattgtattaaacgctgactggagtg,SEQ ID NO:180:tacagtgggtctttcatagactattgggctgaagg,SEQ ID NO:181:actgctatcgagcgtgtttttatgtggagctaata,SEQ ID NO:182:tgggaaacccaaggagggtaaagtgtggtggacca,SEQ ID NO:183:aatagtatagtatcgatgtgttccagtacagaattcctg,SEQ ID NO:184:acaatggaactggcctgacggggctaaaatagagt,SEQ ID NO:185:attggagtaaaaatgatgaaagtactcctatttatgaatc,SEQ ID NO:186:tctgctggaattgaagggtttgagccatactgtat,SEQ ID NO:187:acagttcctcaaatagcaactgtacgaaatacaattgga,SEQ ID NO:188:gattacccttcaacaccagagaggtgctttgatga,SEQ ID NO:189:tagaggaagaaccagaggatgttgatggcccaact,SEQ ID NO:190:aatagtattaagggacatgaataacaaagatgcaaggcaa,SEQ ID NO:191:gataaaggaggaagtaaacactcagaaagaagggaa,SEQ ID NO:192:ccagcgtccgtagaaatgaagggaaagaaacttcc,SEQ ID NO:193:ttgattttgctccaagcaacatagcaccaattggg,SEQ ID NO:194:aaatccaatctatttgtcaccatgtattcctaactttgat,SEQ ID NO:195:aaacgtctgggaagcaacgatgtatcatcatcgtg,SEQ ID NO:196:gcaactttgacaaagacaatgaattgcaactgttttcaaa,SEQ ID NO:197:acaatttggtgccatccaaatccttcacgtatgag,SEQ ID NO:198:tgagctatgcatttgttttgtattgcagaaatactaagaa.

The above probe combinations are probes based on the involvement of genes encoding key surface proteins (hemagglutinin HA and neuraminidase NA) in influenza a viruses of each subtype.

In designing such probe combinations, the HA and NA proteins are faced with a high degree of sequence and structural variation between the different subtypes, and even within the same subtype, viruses may produce new variants due to antigen drift. Thus, in the development of the above-described probe combinations, based on the above-described problems, the inventors developed a variety of probe sequences (e.g., SEQ ID NOs:1-198, described above) to achieve specific capture of viruses of different subtypes. Moreover, even if the virus is mutated, the probe combination can widely identify influenza A viruses of different subtypes. In the design, the inventor also ensures that structures such as dimers are not formed among the probes, and the capturing efficiency is influenced. With such a combination of probes, accurate detection of one or more influenza a viruses that may be present in a sample can be achieved.

Although not all HA and NA combinations are found in nature, certain combinations may not form stable virus subspecies for biological reasons. However, in theory, 16 times 11, i.e., 16×9=144 different combinations can be identified using the above probe combinations. This is also one of the bright spots of the probe combination, enabling the identification of new combinations or gene reassortment, e.g. the combination of H7N 9: the two gene segments of the virus are derived from avian H7N9 virus which is subsequently transferred from wild birds to poultry and subjected to a second reconstitution with H9N2 virus originally circulating in poultry in the specific area, ultimately resulting in H7N9 virus which infects humans. This procedure reveals that the production of novel influenza viruses may be the result of a "multiple reassortment in multiple hosts" and that good identification tracking can be achieved by the above-described probe combinations.

The probes in the probe combination are short probes, and the probes can interact with each other (such as complementary pairing of base sequences at the tail ends of two continuous probes to form a side wall), so that the molecular acting force between the probes and the target can be improved, the combination stability is further improved, the combination efficiency between the probes can be greatly improved, and the capture efficiency of the probes to low-copy pathogens is greatly improved.

When capturing influenza a virus in a sample using the above probe combination, there is a possibility that a single probe may bind to multiple virus subtypes because of the sequence of homology between the different virus subtypes. Thus, accurate typing of the viral subtype is achieved by combinatorial analysis of specific probes detected from the captured sample after capture.

By using the probe combination, the full-length capturing of all influenza A subtypes can be realized in a single day due to the rapid liquid phase hybridization capturing of the matched mu Caler, and the rapid capturing effect is difficult to realize by using the prior art. And by utilizing the probe combination, all known and potential subtypes of influenza A can be covered with high sensitivity and high specificity, and a comprehensive solution is provided, so that the method has important significance in disease monitoring and prevention, and the technical advantage is far superior to RT-qPCR.

In a preferred embodiment, the probes in the probe set each comprise a biomarker thereon.

By using the biomarker, after the probe is hybridized with the target sequence to form a probe-target fragment complex, enrichment and purification of the complex can be realized by using the magnetic beads capable of being combined with the biomarker, so that the subsequent construction of a sequencing library of the target fragment is facilitated.

In a preferred embodiment, the biomarker comprises biotin.

In a second exemplary embodiment of the present application, a kit for detecting influenza a virus is provided, the kit comprising the above-described probe combination for detecting influenza a virus.

In a preferred embodiment, the kit further comprises any one or more of the following: hybridization reaction solution, elution buffer, streptavidin magnetic beads, nucleic acid purification magnetic beads, reverse transcription buffer, PCR buffer, reverse transcriptase, DNA polymerase, end repair enzyme, end repair reaction buffer, molecular tag-containing linker, library amplification primer, linker blocker, DNA blocker, hybridization enhancer or capture library PCR primer.

By using the kit, not only the capture of genes encoding HA and NA in influenza A virus can be realized, but also the operations including, but not limited to, reverse transcription of RNA genetic information of influenza A virus into cDNA, capturing and purifying target fragments in the cDNA, library construction of target fragments obtained by capturing and the like can be realized, so that the whole-flow operation from biological samples to high-throughput sequencing library construction can be realized.

In a third exemplary embodiment of the present application, there is provided a method for detecting influenza a virus, the method comprising: a) Extracting RNA in a sample and carrying out reverse transcription to obtain a cDNA library; b) Mixing and hybridizing the probe combination for detecting the influenza A virus or the probe combination in the kit for detecting the influenza A virus with a cDNA library to obtain a probe-target fragment complex; c) Performing PCR amplification on the probe-target fragment complex to obtain a capture library; d) Sequencing the capture library to detect influenza A virus in the sample.

In the detection method, based on mu Caler rapid liquid phase hybridization capture, the typing and identification work of influenza A virus is realized on an NGS platform. The detection method is shown in FIG. 1. The method can capture 16 subtypes of HA and 11 subtypes of NA of influenza A virus in one experimental flow, so that identification of various combined subtypes of influenza A virus is realized in one high-throughput sequencing reaction, the time required in detection is reduced, the timeliness is improved, and the detection cost is reduced.

In a preferred embodiment, the influenza a virus consists of an HA subtype selected from any one or more of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16; the NA subtype is selected from any one or more of N1, N2, N3, N4, N5, N6, N7, N8 or N9.

In a preferred embodiment, the influenza a virus comprises any one or more of H1N1, H3N2, H3N8, H5N1, H9N2, or H7N 9.

In a preferred embodiment, the above-described detection method is a detection method for non-diagnostic or therapeutic purposes.

In a preferred embodiment, b) comprises: combining and mixing the cDNA library with a probe to obtain a hybridization system; placing the hybridization system at 55-65 deg.C for hybridization for 0.5-2 hr; after the hybridization reaction is finished, capturing and purifying the probe-target fragment complex in the hybridization system by using magnetic beads to obtain the probe-target fragment complex.

In the specific test of the application, firstly, randomly selecting a plurality of samples infected with single subtype influenza, carrying out basic performance test on the probe, and simultaneously assembling the detected gene fragments for evaluating genome coverage and coverage depth of the probe on different subtypes; then, performing mixed test analysis on the sample infected by the single subtype influenza to further evaluate the performance of the designed full-length probe in identifying the sample infected by the mixed influenza virus; finally, samples with pathogen load gradients are selected to evaluate the detection limit of the flow and the scheme.

The advantageous effects of the present application will be explained in further detail below in connection with specific examples.

Example 1

Detection rate of influenza A viruses of different subtypes

According to the designed and synthesized full-length probes of HA and NA, randomly selected influenza samples are identified, and an experimental scheme is designed as follows based on a mu Caler cube rapid capture hybridization system.

Sample information is shown in table 1.

TABLE 1

。

Where "/" means "sum".

1. Nucleic acid extraction

2. Library construction

Library construction was performed by NadPrep mer Total RNA-To-DNA Module library construction kit, as follows:

RNA fragmentation & random primer binding (class B RNA)

1.1, Taking out the Random Primer, naturally melting on ice, uniformly mixing, and performing instantaneous centrifugation for standby.

1.2 The reaction system was formulated in a 0.2 mL PCR tube placed on ice according to the table below, as shown in Table 2.

TABLE 2

。

Note that: if the RNA content is less than 5. Mu.L, the RNA can be supplemented to 5. Mu.L by using Nuclease FREE WATER (no Nuclease water).

1.3, Uniformly mixing, and instantly centrifuging to enable all reaction liquid to be placed at the bottom of the PCR tube.

1.4, Placing the PCR tube into a PCR instrument, and starting the procedure shown in Table 3 below:

TABLE 3 Table 3

。

1.5, Placing the unused reagent back to-25 to-15 ℃ for preservation.

The present specification is divided into two classes according to the quality of RNA samples: full or partially full class a RNAs (cellular, general tissue origin RIN > 7, 3 < RIN < 7; ffpe tissue origin DV 200 > 50%) and highly degraded class B RNAs (cellular, general tissue origin RIN < 3; ffpe tissue origin DV 200 < 50%).

One strand synthesis of cDNA (class B RNA)

2.1, Taking out 1 st Strand Synth. Buffer and 1 st Strand Synth. Enzyme, placing on ice, naturally melting, mixing uniformly, and instantaneous centrifuging for standby.

2.2 Preparation of the reaction system in a 0.2 mL PCR tube placed on ice according to Table 4 below:

TABLE 4 Table 4

。

2.3, Uniformly mixing, and instantly centrifuging to enable all reaction liquid to be placed at the bottom of the PCR tube.

2.4, Placing the PCR tube into a PCR instrument, and starting the procedure shown in Table 5 below:

TABLE 5

。

And 2.5, placing the unused reagent back to-25 to-15 ℃ for preservation.

Two-strand synthesis of cDNA

3.1, Taking out 2nd Strand Synth. Buffer and 2nd Strand Synth. Enzyme, putting on ice to melt naturally, mixing uniformly, and centrifuging instantaneously for standby.

3.2 Preparation of the reaction system in a 0.2 mL PCR tube placed on ice according to Table 6 below:

TABLE 6

。

And 3.3, uniformly mixing, and instantly centrifuging to enable all reaction liquid to be placed at the bottom of the PCR tube.

3.4, The reaction procedure shown in Table 7 below was started on a PCR apparatus, and the reaction tube was put into the PCR apparatus when the isothermal temperature was stabilized to 16 ℃.

TABLE 7

。

4. Purification of double-stranded DNA

4.1, Adding NadPrep ℃ SP Beads of 90 mu L into the cDNA two-chain synthesis product obtained by the preparation, uniformly mixing, and incubating at 25 ℃ for 10 min.

4.2, Placing the PCR tube on a magnetic rack after instantaneous centrifugation for 5 min to completely clarify the liquid, and sucking and discarding the supernatant by using a pipette.

Note that: the magnetic rack must be clear completely, and the placement time of different brands must be adjusted.

4.3, Slowly adding 150 mu L of 80% ethanol along the side wall of the PCR tube, taking care of not disturbing the magnetic beads, standing for 30 sec, and sucking and discarding the supernatant by using a pipette.

4.4, Repeating the step 4.3 once.

4.5, Placing the PCR tube on a magnetic rack after instantaneous centrifugation, and removing a small amount of residual ethanol by using a 10 mu L suction head, taking care of not sucking the residual ethanol to the magnetic beads.

4.6, Opening the tube cover of the PCR tube, and standing at room temperature for about 5min until the ethanol is completely volatilized.

Note that: it is not excessively dried, otherwise the yield is lowered.

4.7, Removing the PCR tube, adding 45 mu L of nucleic FREE WATER into the PCR tube, suspending the magnetic beads uniformly by using a pipette, and incubating at 25 ℃ for 2 min.

4.8, The PCR tube was centrifuged instantaneously and placed on a magnetic rack for 2 min a until the liquid was completely clarified, and the supernatant was carefully transferred to a new 0.2 mL PCR tube for storage using a pipette, taking care not to suck the beads.

4.9, Quantifying double-stranded DNA using a nucleic acid fluorescence quantitative instrument.

4.10, The double-stranded DNA product should avoid repeated freeze thawing, and can be preserved for one week at minus 25 to minus 15 ℃ or preserved for a long period at minus 80 ℃.

5. End repair & addition A

And 5.1, taking out END REPAIR & A-Tailing Buffer, melting at normal temperature, mixing uniformly, and placing on ice for standby.

And 5.2, taking out END REPAIR & A-Tailing Enzyme, placing on ice for natural melting, uniformly mixing, and performing instantaneous centrifugation for standby.

5.3 Preparation of the reaction system in a 0.2 mL PCR tube placed on ice according to Table 8 below:

TABLE 8

。

Note that: if the DNA is less than 40. Mu.L, the nucleotide FREE WATER can be used to make up to 40. Mu.L.

And 5.4, uniformly mixing, and instantly centrifuging to enable all reaction liquid to be placed at the bottom of the PCR tube.

5.5, Starting the reaction procedure shown in the following Table 9 on a PCR apparatus, and placing the reaction tube into the PCR apparatus when the isothermal temperature is stabilized to 20 ℃):

TABLE 9

。

Note that: the hot cap was set to 70 ℃ during this procedure.

And 5.6, placing the unused reagent back to ‒ - ‒ ℃ for preservation.

6. Joint connection

And 6.1, taking out the Ligation Buffer, melting at normal temperature, uniformly mixing, and placing on ice for standby.

Note that: the Ligation Buffer is very viscous, and the pipette should aspirate and release slowly and smoothly to ensure accurate volume.

And 6.2, taking out DNA LIGASE, naturally melting on ice, uniformly mixing, and performing instantaneous centrifugation for standby.

6.3, Taking out the PCR reaction tube from the PCR instrument, placing the PCR reaction tube on ice, and preparing a reaction system according to the following table 10:

Table 10

。

Note 1: to avoid self-connection of the linker, nadPrep. Universal Stubby Adapter should be added to the bottom of the PCR tube first, followed by addition of Ligation Buffer and DNA LIGASE.

Note 2: in the multi-sample operation, in order to avoid the self-connection of the joint caused by slow operation, the mixed solution of the Ligation Buffer and DNA LIGASE should be pre-configured proportionally.

And 6.4, uniformly mixing, and instantly centrifuging to enable all reaction liquid to be placed at the bottom of the PCR tube.

6.5, Starting the reaction procedure shown in the following Table 11 on a PCR apparatus, and placing the reaction tube into the PCR apparatus when the isothermal temperature is stabilized to 20 ℃):

TABLE 11

。

Note that: this procedure does not require a hot cap.

And 6.6, placing the unused reagent back to-25 to-15 ℃ for preservation.

6.7 Purification of ligation products

6.7.1, Taking out NadPrep cube SP Beads in advance, mixing uniformly by vortex, and balancing at room temperature for 30 and min.

6.7.2, Adding NadPrep ℃ SP Beads with a volume of 40 mu L into the prepared ligation reaction product, uniformly mixing, and incubating at 25 ℃ for 5-10 min.

6.7.3, Placing the PCR tube on a magnetic rack after instantaneous centrifugation for 5: 5min until the liquid is completely clarified, and sucking and discarding the supernatant by using a pipette.

Note that: the magnetic rack must be clear completely, and the placement time of different brands must be adjusted.

6.7.4, Slowly adding 150 mu L of 80% ethanol along the side wall of the PCR tube, taking care of not disturbing the magnetic beads, standing for 30: 30 sec, and sucking and discarding the supernatant by using a pipette.

6.7.5, Repeating the step 6.7.4 once.

6.7.6, Placing the PCR tube on a magnetic rack after instantaneous centrifugation, and removing a small amount of residual ethanol by using a 10 mu L suction head, taking care of not sucking the PCR tube to the magnetic beads.

6.7.7, Opening the PCR tube cap, and standing at room temperature for about 5min a until the ethanol is completely volatilized.

Note that: it is not excessively dried, otherwise the yield is lowered.

6.7.8, Removing the PCR tube, adding 20 mu L of nucleic FREE WATER into the PCR tube, and carrying out PCR amplification with magnetic beads.

PCR amplification

8.1, Taking out 2X HiFi PCR Master Mix and NadPrep ℃ Universal UDI-Index Primer Mix, placing the mixture on ice for natural melting, uniformly mixing, and carrying out instantaneous centrifugation for standby.

8.2 Preparation of the reaction system (addition from top to bottom) in a 0.2 mL PCR tube placed on ice according to Table 12 below:

table 12

。

8.3 Placing the PCR tube in a PCR instrument and starting the procedure shown in Table 13 below:

TABLE 13

。

8.4, Placing the unused reagent back to ‒ - ‒ ℃ for preservation.

9. Amplified library purification

9.1, Adding NadPrep cube SP Beads of 50 mu L into the PCR tube of the amplification reaction product obtained by the preparation, uniformly mixing, and incubating at 25 ℃ for 5 ‒ and min.

9.2, The PCR tube is placed on a magnetic rack after instantaneous centrifugation

5 Min to complete clarification of the liquid, pipetting the supernatant using a pipette.

Note that: the magnetic rack must be clear completely, and the placement time of different brands must be adjusted.

And 9.3, slowly adding 150 mu L of 80% ethanol along the side wall of the PCR tube, taking care of not disturbing the magnetic beads, standing for 30 sec, and sucking and discarding the supernatant by using a pipette.

9.4, Repeating the step 9.3 once.

9.5, Placing the PCR tube on a magnetic rack after instantaneous centrifugation, and removing a small amount of residual ethanol by using a 10 mu L suction head, taking care of not sucking the residual ethanol to the magnetic beads.

9.6, Opening the tube cover of the PCR tube, and standing at room temperature for about 5min until the ethanol is completely volatilized.

Note that: it is not excessively dried, otherwise the yield is lowered.

And 9.7, removing the PCR tube, adding 20 mu L TE Solution into the PCR tube, and uniformly suspending the magnetic beads by using a pipettor, and incubating at 25 ℃ for 2 min.

9.8, After the PCR tube was transiently centrifuged, the solution was placed on a magnetic rack for 2 min a until the solution was completely clarified, and the supernatant was carefully transferred to a new 0.2 mL PCR tube for storage using a pipette, taking care not to suck the beads.

3. Hybrid capture

Hybrid Capture is performed by a mu Caler cube Hybrid Capture kit of REAGENTS V2, and the method comprises the following steps:

The preparation is shown in table 14.

TABLE 14

。

Note that: if mu Hyb #1 and Wash Buffer A Pro are used/stored in a split-charging mode, the original tube/bottle is heated until the crystals are completely melted, then split-charging operation is performed, and direct split charging is forbidden. In addition, it was ensured that all reagent components had been sufficiently thawed and vortexed well before use.

1. Library hybridization

1.1, Preparation of hybridization reaction System according to Table 15:

TABLE 15

。

Note that: * To maintain library complexity, it is recommended that 50% or more of the total amount of each constructed library be used for hybridization. If the total library volume is > 18. Mu.L, the library can be concentrated. The number of pre-libraries contained in the total library is optionally 1, 2, 3,4, 5 or 6.

1.2, Mixing uniformly by vortex, hybridizing the mixed solution of the hybridization reaction by more than 10 sec, and collecting the mixed solution of the hybridization reaction to the bottom of a PCR tube after instantaneous centrifugation (avoiding generating bubbles).

1.3, Placing the PCR tube into a PCR instrument, and starting the reaction procedure shown in the following Table 16:

Table 16

。

2. Magnetic bead cleaning

2.1, STREPTAVIDIN BEADS vortex 15 sec to ensure complete mixing.

2.2, Taking n multiplied by 25 mu L STREPTAVIDIN Beads and mixing and cleaning in a 0.2 mL centrifuge tube (n is the total capture reaction number and n is less than or equal to 5).

Note that: wash Buffer A Pro is placed back into 50-60 ℃ for heating.

2.3, Placing STREPTAVIDIN BEADS on a magnetic rack for standing for about 2 min, and discarding the supernatant by using a pipette after the liquid is completely clarified.

2.4, Removing the centrifuge tube from the magnetic rack, adding 150 mu L of preheated Wash Buffer A Pro, and gently blowing and sucking to mix for more than 10 times.

2.5, Placing the centrifuge tube on a magnetic rack for standing for about 2 min, and discarding the supernatant by using a pipette after the liquid is completely clarified.

2.6, Repeating steps 2.4 and 2.5 once.

Note that: when n is more than 5, the mixture is divided into multiple pipes for mixed cleaning.

2.7, The centrifuge tube was centrifuged instantaneously and then placed on a magnetic rack to stand for 10 sec. Mu.L, and Wash Buffer A Pro at the bottom of the tube was completely discarded by a 10. Mu.L pipette.

2.8, Taking n multiplied by 9 mu L mu Hyb#1 to resuspend STREPTAVIDIN BEADS, gently flushing and mixing for more than 10 times.

3. Magnetic bead capture

And 3.1, after hybridization reaction, entering a capturing link, and maintaining the running state of the PCR instrument.

3.2, Taking 8 mu L of resuspended STREPTAVIDIN BEADS to be immediately added into each hybridization reaction liquid under the state that the PCR tube is kept in a PCR instrument, and gently blowing and sucking the mixture for more than 10 times.

Incubation at 60℃at 3.3 for 10 min.

And 3.4, after incubation, taking the PCR tube from the PCR instrument, placing the PCR tube on a magnetic rack, standing for 2min, and discarding the supernatant (removing the supernatant as much as possible) by using a pipette after the liquid is completely clarified.

4. Elution

4.1, Removing the PCR tube from the magnetic rack, adding 150 mu L of preheated Wash Buffer A Pro, gently blowing and sucking, and mixing for more than 10 times (avoiding generating bubbles).

4.2, Placing the PCR tube on a magnetic rack for standing for 2 min, and discarding the supernatant by using a pipette after the liquid is completely clarified.

4.3, Removing the PCR tube from the magnetic frame, adding 100 mu L of preheated Wash Buffer A Pro, gently blowing and sucking, uniformly mixing for more than 10 times, and transferring the reaction solution into a new PCR tube.

Note that: this step avoids the creation of bubbles as much as possible, which could potentially lead to a reduced mid-target rate when contacting the tube cap.

4.4, Placing the PCR tube into a PCR instrument, and incubating at 60 ℃ for 3 min.

And 4.5, after incubation, taking the PCR tube from the PCR instrument, placing the PCR tube on a magnetic rack, standing for 2 min, and discarding the supernatant by using a pipette after the liquid is completely clarified.

4.6, 150 Mu L of Wash Buffer B placed at room temperature is added into the PCR tube, and the mixture is gently blown and sucked for more than 10 times.

4.7, Placing the PCR tube on a magnetic rack for standing for 2 min, and discarding the supernatant by using a pipette after the liquid is completely clarified.

4.8, Placing the PCR tube on a magnetic rack after instantaneous centrifugation, and removing a small amount of residual liquid by using a 10 mu L suction head, taking care of not sucking the residual liquid to the magnetic beads.

4.9, 22.5. Mu.L Nuclease FREE WATER was added and the beads were gently flushed more than 10 times.

The optional steps are as follows: the resuspended magnetic beads are all transferred to a new PCR tube, so that the nonspecific capture can be reduced, and the medium target rate can be improved

5. Hybrid Capture library PCR amplification

And 5.1, taking out the 2X PCR Master Mix and the Primer Mix, naturally thawing on ice, gently mixing uniformly by using a pipette or a vortex mixer, and performing instantaneous centrifugation for later use.

5.2 The reaction system was placed in a centrifuge tube placed on ice according to the system of Table 17.

TABLE 17

。

5.3 Placing the PCR tube in a PCR instrument and starting the procedure shown in Table 18 below.

TABLE 18

。

And (3) detecting the concentration by using the Qubit immediately after amplification is finished, >2 ng/MuL, and avoiding excessive amplification.

6. Library purification and quantification

And 6.1, taking out the PCR tube after the PCR amplification is finished, placing the PCR tube on a magnetic rack for 2 min, and transferring the supernatant into 1 new centrifuge tube by using a pipette after the supernatant is completely clarified.

And 6.2, adding NanoPrep SP Beads mu L of the solution into the PCR tube, uniformly mixing the solution by using a pipette or a vortex mixer, and incubating the solution at 25 ℃ for 5 min.

And 6.3, after the PCR tube is subjected to short centrifugation, placing the PCR tube on a magnetic rack for 5 min, and completely clarifying the supernatant, and sucking the abandoned supernatant by using a pipette.

6.4, Slowly adding 150 mu L of 80% ethanol along the side wall of the PCR tube, taking care of not disturbing magnetic beads, standing for 30 s, and sucking the abandoned supernatant by using a pipette.

And 6.5, repeating the step 6.4 once.

6.6, The PCR tube is centrifuged briefly and placed on a magnetic rack, and a small amount of residual ethanol is removed by using a 10 mu L suction head, and the magnetic beads are not sucked.

And 6.7, placing the PCR tube back on a magnetic rack, standing at room temperature for 5min a until the ethanol is completely volatilized (the DNA is damaged and difficult to redissolve due to the fact that the ethanol is not excessively dried).

6.8, Adding 15 mu L TE Solution into the PCR tube, suspending the magnetic beads uniformly by using a pipette, and incubating at 25 ℃ for 2 min.

6.9, After the PCR tube is briefly centrifuged, the PCR tube is placed on a magnetic rack for 2 min, the supernatant is completely clarified, and a pipette is used for carefully transferring the supernatant into a new centrifuge tube for preservation, and the supernatant is not sucked to the magnetic beads.

Quantification of the library was performed using Qubit, concentration >2 ng/μl.

The test results are shown in table 19:

TABLE 19

。

The application of the method in the process compares qPCR results of the traditional detection method for disease control, the detection results show higher consistency (detailed detection frequency is shown in figure 2), and the system detection results are basically consistent with gold standards for 4 influenza viruses of different subtypes, namely H3N2, H3N8, H5N1 and H7N 9.

The HA（1-16）：H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16,NA（1-11）：N1、N2、N3、N4、N5、N6、N7、N8、N9、N10、N11 full-length mixing tube probe designed in the present invention, which identifies the performance of different influenza a virus subtypes, randomly selected influenza a infection samples (qPCR identification results): the consistency rate of the main abundance virus subtype detected by the technical process and qPCR results of H3N2, H5N1, H3N8 and H7N9 is 100%, and the detection rates of the virus subtypes of different samples are 91.5%, 78.8%, 66.5% and 87.2%, respectively.

Example 2

Coverage and coverage depth of HA and NA genes of different subtype influenza viruses

Based on the results of 4 samples randomly selected in the embodiment 2, the genome coverage and coverage depth of influenza viruses with high detection frequency and dominant abundance in the samples are analyzed in detail through the letter end, and the detailed results are shown in fig. 3A, 3B, 3C, 3D and 3E. From the figure, the main virus subtypes detected by 4 samples can be seen, and the genome coverage rate is close to 100% after the gene assembly, so that the full coverage of HA and NA genes of all subtype influenza viruses can be realized.

FIGS. 3A-3D show coverage and depth of coverage for HA and NA genes of different subtypes of influenza virus, and FIG. 3E shows a population of ectopic sites, from which it can be seen that the major viral subtypes detected by 4 samples, after gene assembly, have genome coverage approaching 100%, with the exception of the H3 subtype of sample LQ12 (H3), the remaining depth of coverage being relatively uniform, which may be related to the sample itself, because in samples ML24040803-IV the depth of coverage for H3 is relatively uniform; FIG. 3E shows that some bases have a polymorphism, suggesting that the liquid phase hybridization capture technique has an ability to track viral variation over Sanger sequencing.

This example demonstrates that the HA（1-16）：H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16 ,NA（1-11）：N1、N2、N3、N4、N5、N6、N7、N8、N9、N10、N11 full-length mixed tube probe designed by the present invention has expected genome coverage for different subtypes and relatively uniform coverage depth, and shows that the probe designed by the technical process has superior performance and can meet the requirements of different subtype identification. Meanwhile, in the genome assembly process, as shown in fig. 3E, there is a display of a variable site group, which is also an advantage of the process, and the presentation of base polymorphism suggests that the liquid phase hybridization capture technology has the capability of virus mutation tracking.

Example 3

Sample detection results of multiple mixed infection influenza strains

To further evaluate whether the method of the present invention can be applied to clinical mixed infection with multiple influenza virus subtypes or mixed infection with common human influenza and avian influenza, samples containing multiple influenza virus subtypes were mixed and tested, and samples were selected as in example 2, and the test results are shown in table 20 below.

Table 20

。

It can be seen from table 20 that the higher frequency of detection of the virus subtype was consistent with the highest target abundance virus subtype in the initially selected sample, and that the higher abundance identified serotype combination and mixed infection samples achieved 100% in reference subtype concordance, with a total influenza detection rate of 45.7%, as shown in fig. 4.

Fig. 4 and table 20 show samples of 16 HA and 9 NA virus subtypes of mixed tube probes versus mixed infection influenza strains: the 4 subtypes of H3N2, H5N1, H3N8 and H7N9 are mixed, the consistency of the identified high-abundance serotype combination and the reference subtype of the mixed infection sample reaches 100%, and the results of detection conditions and detection frequencies of different serotypes are shown in figure 4.

This example demonstrates that the mixed tube probe of 16 HA and 9 NA virus subtypes of the present invention HAs better discrimination ability for samples of mixed-infection influenza strains.

Example 4

Detection of influenza samples of different viral loads

In order to further evaluate the advantages of the present invention, in this embodiment, samples with pathogen load gradients were selected to evaluate the detection limits of the present invention flow and protocol.

RNA nucleic acid library construction, nucleic acid extracted from pharyngeal swabs matched NadPrep Total RNA-To-DNA Module library construction kit, RNA library construction, library construction and hybridization capture using samples containing the following pathogens, sample selection is shown in table 21.

Table 21

。

All samples are influenza A virus infection samples, the same subtype infection is carried out, the pathogen load has gradient change, and the detection limit of the technical process of the invention is tested: species were detected, detection rate and clinical identification result consistency.

Step one: library construction

See example 1

Step two: hybrid capture

See example 1

The test results are shown in table 22:

Table 22

。

Note that: HA and NA subtype detection results only showed the highest ready ratio of typing results.

The above were downsample down to 0.02G, clinical sample types: a pharyngeal swab. The coverage and depth of coverage are shown in detail in fig. 5 and 6A, 6B, and 6C.

FIG. 5 shows the spread of the detection limit of the system of the invention on influenza: the test using different pathogen load throat swab clinical sample types shows that the HA genome coverage can reach 100% when the influenza virus full-length assembly detection limit is H1N1 (Ct=34)/H3N 2 (Ct=31)/H7N 9 (Ct=34); the detection limit of the influenza virus identified species is H1N1 (Ct=36)/H3N 2 (Ct > 31)/H7N 9 (Ct=37) overall performance superior to RT-gPCR.

Fig. 6A-6C show analysis of the sequencing depth of samples with HA genome coverage up to 100% detected in sample detection of different viral loads by the system of the present invention.

In the analysis of HA and NA full-length coverage, when H1N1 (Ct=34)/H3N 2 (Ct=31)/H7N 9 (Ct=34), the HA genome coverage can reach 100 percent.

The detection limit of the influenza virus identified species is H1N1 (Ct=36)/H3N 2 (Ct > 31)/H7N 9 (Ct=37), and the overall performance is superior to that of RT-gPCR.

For samples with negative 2 Ct values in table 22: H3N2 ML240423H3-7_S57 which is not detected by RT-qPCR can be detected in the current flow, and the genome coverage is more than 50%; no RT-qPCR of H7N9 ML240423H7-9_S71 sample was detected, but the NA gene coverage and HA gene coverage were significantly different, and the coverage analysis was not participated here.

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the invention is designed based on mu Caler targeting hybridization capture flow, and is applied to detection of various subtypes of acute influenza by utilizing the advantage of rapid hybridization. Breaks the limitation of the traditional detection technology which can detect only a limited number of strains and consumes time and labor in the prior art.

Compared with the prior art, the invention has at least the following advantages:

1. The method has more timeliness: compared with the traditional liquid phase hybridization capture flow, the method can complete the database construction and capture on-machine sequencing of pathogen samples in a single day, and meets the timeliness of epidemiological pathogen detection;

2. Simpler operation/lower cost: one sample can be used for completing identification of all influenza A virus subtypes by only establishing one library, and compared with the prior art, such as serological identification, the clinical accessibility is stronger;

3. the variation and human infection of avian viruses can be tracked: the probe has certain fault tolerance, can track the virus variation condition, and can discover new subtype and/or human virus strain infecting birds in time due to the comprehensive subtype of the designed influenza virus.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. A probe combination for detecting influenza A virus, characterized in that the probe combination comprises a probe for capturing HA gene and a probe for capturing NA gene,

The probe combinations include the sequences as set forth in SEQ ID NOs:1-198, and a combination of nucleic acid sequences as set forth in seq id no.

2. The probe assembly of claim 1, wherein the probes in the probe assembly each comprise a biomarker.

3. The probe combination of claim 2, wherein the biomarker comprises biotin.

4. A kit for detecting influenza a virus, comprising a probe combination according to any one of claims 1-3 for detecting influenza a virus.

5. The kit of claim 4, further comprising any one or more of the following: hybridization reaction solution, elution buffer, streptavidin magnetic beads, nucleic acid purification magnetic beads, reverse transcription buffer, PCR buffer, reverse transcriptase, DNA polymerase, end repair enzyme, end repair reaction buffer, molecular tag-containing linker, library amplification primer, linker blocker, DNA blocker, hybridization enhancer or capture library PCR primer.

6. A method for detecting influenza a virus, the method comprising:

a) Extracting RNA in a sample and carrying out reverse transcription to obtain a cDNA library;

b) Mixing and hybridizing the probe combination with the cDNA library to obtain a probe-target fragment complex; the probe combination is the probe combination for detecting influenza a virus according to any one of claims 1 to 3, or the probe combination in the kit for detecting influenza a virus according to claim 4 or 5;

c) Performing PCR amplification on the probe-target fragment complex to obtain a capture library;

d) Sequencing the capture library to effect detection of the influenza a virus in the sample;

The detection method is a detection method for non-diagnostic or therapeutic purposes.

7. The method according to claim 6, wherein the influenza A virus consists of subtype HA and subtype NA,

The HA subtype is selected from any one or more of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16;

The NA subtype is selected from any one or more of N1, N2, N3, N4, N5, N6, N7, N8 or N9.

8. The method of claim 7, wherein the influenza a virus comprises any one or more of H1N1, H3N2, H3N8, H5N1, H9N2, or H7N 9.

9. The detection method according to any one of claims 6 to 8, wherein b) comprises:

combining and mixing the cDNA library with the probe to obtain a hybridization system;

Placing the hybridization system at 55-65 ℃ for hybridization for 0.5-2 hours;

After the hybridization reaction is finished, capturing and purifying the probe-target fragment complex in the hybridization system by using magnetic beads to obtain the probe-target fragment complex.