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CN-121992153-A - Primer group, method and application for sequencing whole genome of human metapneumovirus

CN121992153ACN 121992153 ACN121992153 ACN 121992153ACN-121992153-A

Abstract

The invention discloses a primer group, a method and application for sequencing a whole genome of a human metapneumovirus. The invention designs 2 groups of specific primer pools aiming at metapneumovirus, contains 21 primers in total, can effectively amplify metapneumovirus A type and metapneumovirus B type, has high sensitivity and is suitable for multi-platform detection, and the invention provides a new direction for metapneumovirus detection, typing, tracing and evolution analysis.

Inventors

  • HE XIAOZHOU
  • MA XUEJUN
  • LI FAN
  • SU QIUDONG
  • REN JUNKAI

Assignees

  • 中国疾病预防控制中心病毒病预防控制所

Dates

Publication Date
20260508
Application Date
20260227

Claims (10)

  1. 1. A primer combination for metapneumovirus whole genome enrichment, characterized in that the primer combination comprises a primer pool a and a primer pool B; preferably, the nucleotide sequence of the primer pool A is shown as SEQ ID NO. 1-9; preferably, the nucleotide sequence of the primer pool B is shown as SEQ ID NO. 10-21; preferably, the primer combination further comprises a nucleotide sequence as shown in SEQ ID NOS.74-75 or SEQ ID NOS.76-139.
  2. 2. The primer combination of claim 1, wherein the primer combination comprises a degenerate base; Preferably, the degenerate base comprises R, W, H or Y, wherein R comprises A or G, W comprises A or T, H comprises A, C or T, and Y comprises C or T.
  3. 3. A method for amplifying metapneumovirus whole genome sequence, comprising performing an amplification reaction using the primer combination of claim 1 or 2.
  4. 4. The method of claim 3, wherein the method comprises performing a first round of amplification using the metapneumovirus cDNA as a template using the primer pool A and the primer pool B, respectively, and performing a second round of amplification using the primers shown in SEQ ID NOS 74-75 or SEQ ID NOS 76-139 as templates; preferably, the amplification method further comprises extracting metapneumovirus nucleic acid from the sample to be tested; preferably, the amplification method further comprises reverse transcription of the viral nucleic acid to obtain cDNA; preferably, the amplification method further comprises the step of purifying and quantifying the amplified product; Preferably, the amplification method further comprises the step of sequencing by constructing a library using the second round of amplification products.
  5. 5. The amplification method of claim 3, wherein the amplification reaction is performed by a method comprising LCR, NASBA, SDA, TMA, bDNA, PCR; Preferably, the amplification reaction is performed by a PCR method; preferably, the first round of amplification is performed with a system of PRIMESTAR MAX. Mu.l, 20. Mu.l of primer pool A or primer pool B primers, 5. Mu.l of cDNA template; preferably, the second round amplification system is PRIMESTAR MAX. Mu.l, 5. Mu.l of the primer shown in SEQ ID NO:74-75 or SEQ ID NO:76-77, 5. Mu.l of the first round amplification product, and 15. Mu.l of ddH 2 O; preferably, the reaction conditions for the PCR amplification are (98 ℃ 10 s- > 50 ℃ 15 s- > 72 ℃ 40 s) cycled 5 times, (98 ℃ 10 s- > 55 ℃ 5 s- > 72 ℃ 20 s) cycled 30 times, 72 ℃ 5min cycled 1 time, 4 ℃ Hold.
  6. 6. The method according to claim 4, wherein the sample to be tested comprises a nasopharyngeal swab, a pharyngeal swab, saliva, sputum, and alveolar lavage; preferably, the purification is magnetic bead purification; Preferably, the sequencing comprises second generation sequencing and third generation sequencing.
  7. 7. A product for detecting metapneumovirus or amplifying metapneumovirus whole genome sequence, characterized in that it comprises a primer combination according to claim 1 or 2; Preferably, the product comprises a chip, a nucleic acid membrane strip, a kit.
  8. 8. The product of claim 7, wherein the kit further comprises an acceptable adjuvant; preferably, the acceptable auxiliary agents comprise PCR amplification buffer, amplification enzyme; preferably, the amplification enzyme comprises a DNA polymerase or an RNA polymerase; Preferably, the DNA polymerase comprises PRIMESTAR MAX DNA polymerase, taq DNA polymerase, pfu DNA polymerase, KOD DNA polymerase, Q5 high-fidelity DNA polymerase, Q5 hot-start super-fidelity DNA polymerase, phanta high-fidelity DNA polymerase, VAHTS HIFI DNA polymerase; preferably, the acceptable auxiliary agent further comprises a reverse transcription reaction system; preferably, the reaction system of reverse transcription comprises a primer, a reverse transcription buffer, reverse transcriptase, ribonuclease inhibitor, DTT, dNTPs; Preferably, the primer is a random primer; Preferably, the reverse transcription Buffer comprises M-MLV reverse transcription Buffer, hifair: III REVERSE TRANSCRIPTASE Buffer, hifair: VBuffer and SSIV Buffer; Preferably, the reverse transcriptase comprises an M-MLV RT enzyme, an HIV RT enzyme, an ASLV RT enzyme, an RSV RT enzyme, an AMV RT enzyme, A REV-A RT enzyme, A RAV RT enzyme, A MAV RT enzyme, an SSIV RT enzyme; preferably, the kit further comprises instructions.
  9. 9. Use of a primer combination according to claim 1 or 2 for the preparation of a kit for detecting metapneumovirus or for the preparation of a metapneumovirus whole genome amplified and/or sequenced product; Preferably, the metapneumovirus comprises metapneumovirus type a and/or metapneumovirus type B.
  10. 10. Use of the primer combination of claim 1 or 2 or the product of claim 7 or 8 in metapneumovirus typing, traceability or evolutionary analysis; Preferably, the metapneumovirus comprises metapneumovirus type a and/or metapneumovirus type B.

Description

Primer group, method and application for sequencing whole genome of human metapneumovirus Technical Field The invention belongs to the technical field of biology, and particularly relates to a primer group, a method and application for sequencing a whole genome of a human metapneumovirus. Background Human metapneumovirus (human metapneumo virus, hMPV) is a common human respiratory pathogen, and particles are polymorphic, spherical, fibrous under electron microscopy. hMPV belongs to the pneumoviridae family, genus metapneumovirus, enveloped single-stranded negative-strand RNA viruses. hMPV shares A, B genotypes, contains 8 genes and 9 open reading frames, and encodes proteins mainly including nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), transcription elongation factor (M2)1) RNA synthesis regulator (M2)2) Small hydrophobic surface proteins, adhesion proteins (G), and polymerase (L) subunits. There are three commonly used methods for detecting hMPV, including antigen-antibody detection, virus cell culture and reverse transcription polymerase chain Reaction (RT)PCR). The virus cell culture technique has higher requirements, longer culture time, difficult recognition of cytopathic effect and lower sensitivity. The antigen detection of HMPV is commonly used in clinic by immunofluorescence, immunochromatography and enzyme immunoassay. However, these detection techniques can only identify the pathogen, but cannot analyze the whole genome sequence of the pathogen, and cannot further track and trace the epidemic variation of the virus. It is therefore important to provide a method for whole genome amplification and sequencing of human metapneumoviruses. Disclosure of Invention In order to overcome the defects of the prior art, the invention aims to provide a metapneumovirus whole genome amplification primer and a sequencing method which have high sensitivity and high flux and are suitable for multiple platforms. In order to achieve the above purpose, the present invention adopts the following technical scheme: the first aspect of the invention provides a primer combination for metapneumovirus whole genome enrichment. Further, the primer combination includes a primer pool A and a primer pool B. Further, the nucleotide sequence of the primer pool A is shown as SEQ ID NO. 1-9. Further, the nucleotide sequence of the primer pool B is shown as SEQ ID NO. 10-21. Further, the primer combination also comprises nucleotide sequences shown as SEQ ID NO. 74-75 or SEQ ID NO. 76-139. In the present invention, the primer Pool A and Pool A can be used interchangeably, and similarly, the primer Pool B and Pool B can be used interchangeably. In some embodiments, the primer combination further comprises a primer sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NOS.1-21. Identity/similarity refers to identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, expressed in terms of identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity, with higher percentages being more identical sequences. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high level of sequence identity/similarity when aligned using standard methods. The primers disclosed herein are not limited to the exact sequences shown, as those skilled in the art will recognize that changes may be made to the sequences if desired without significantly affecting the ability of the primers to function. Those skilled in the art will recognize that these ranges of sequence identity are provided for instructional purposes only and that primers that are not within these ranges may be used. Further, the primer combination includes a degenerate base. Further, the degenerate positions include R, W, H or Y, where R includes A or G, W includes A or T, H includes A, C or T, and Y includes C or T. In some embodiments, the degenerate positions of the degenerate sequence comprise R, W, M, Y, K, B, D, H, N, S or V, wherein R comprises a or G, W comprises a or T, M comprises a or C, Y comprises C or T, K comprises G or T, B comprises A, G or T, D comprises A, G or T, H comprises A, C or T, N comprises A, G, C or T, S comprises G or C, and V comprises A, C or G. In some embodiments, at least one of the primer sets may be labeled with a labeling substance. The labeling substance comprises fluorescent label, radioisotope, chemiluminescent molecule and paramagnetic ion. In some embodiments, the primer set includes at least one modified nucleotide. In some embodiments, modified nucleotides include, but are not limited to, 2' -modified nucleotides or 5-methylcytosine. Wherein the 2 '-modified nucleotide comprises but is not limited to 2' -O-methyl modified nucleotide and 2 '-fluoro modified oligonucleotide, the 5-methylcytosine comprises but is not limited to 5-methyl-deoxycytosine, and the 5-methyl-deoxycytosine