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Amplification method for whole genome aiming at different enterovirus serotypes

An enterovirus and whole genome technology is applied in the field of amplification of whole genomes of different enterovirus serotypes. The amplification method is simple and easy, the rapid amplification method, and the effect of fast amplification speed

Inactive Publication Date: 2016-04-27
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can be used to amplify the whole genome of other viruses (such as mumps virus, measles virus, etc., these viruses have only one serotype) to obtain better results. Nucleotide differences are large, and differences between the same serotype are also large. In addition, enteroviruses also commonly have recombination events. It is difficult to amplify the whole genome sequence of enteroviruses by traditional methods, and the amplification time is often as long as 3-6 months, time-consuming and labor-intensive, the workload is very heavy, and the cost is high
[0004] Enteroviruses are pathogens that cause many diseases such as hand, foot and mouth disease, viral encephalitis, myocarditis, etc., and are also RNA viruses, whose genes mutate rapidly. The existing genome amplification methods cannot meet the needs. It is very necessary to improve the intestinal Method for Amplifying the Whole Genome Sequence of Viruses

Method used

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  • Amplification method for whole genome aiming at different enterovirus serotypes
  • Amplification method for whole genome aiming at different enterovirus serotypes
  • Amplification method for whole genome aiming at different enterovirus serotypes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Taking Coxsackievirus B1 (CV-B1) as an example

[0024] (1) Perform RT-PCR on the VP1 region of the viral RNA with enterovirus general primer 222 (5'CICCIGGIGGIAYRWACAT3')-224 (GCIATGYTIGGIACICAYRT), and then use 1% agarose gel for electrophoresis detection at a voltage of 120V, and the detection results Compared with the predicted results, the results are similar (see figure 2 ), after sequencing, use NCBIBLAST (a set of analysis tools for similarity comparison in the protein database or DNA database of the National Center for Biotechnology Information of the United States, and the BLAST program can quickly compare similarity sequences with public databases) to compare, the nucleoside The homology between the acid sequence and Coxsackie virus B1 is 92%. According to the typing standard, it can be judged that the virus is Coxsackie virus B1 (CV-B1). This amplification obtained part of the VP1 in the genome of the virus. the nucleotide sequence of the gene; ...

Embodiment 2

[0042] Example 2: Taking Coxsackievirus B2 (CV-B2) as an example

[0043] (1) Perform RT-PCR on the VP1 region of the viral RNA with enterovirus general primers 222-224, and then use 1% agarose gel for electrophoresis detection at a voltage of 120V. The detection results are compared with the predicted results, and the results are similar ( See figure 2 ), compared with NCBIBLAST after sequencing, the nucleotide sequence has a homology of 92% with CV-B2. According to the typing standard, the virus can be judged as CV-B2, and part of the VP1 in the genome of the virus can be obtained The nucleotide sequence of the gene.

[0044] (2) According to the conserved type of the 5' end and 3' end of the enterovirus genome, and the nucleotide sequence of the part of the VP1 gene obtained in step (1), design primers; divided into two sections, the designed primers are: EV1F-B22R ( About 2400bp), B23F-EV8R (about 5000bp) (see Table 4), using viral RNA as a template, EV1F-B22R (about 24...

Embodiment 3

[0061] Example 3: Taking echovirus type 33 (E-33) as an example

[0062] (1) Perform RT-PCR on the VP1 region of the viral RNA with enterovirus general primers 222-224, and then use 1% agarose gel for electrophoresis detection at a voltage of 120V. The detection results are compared with the predicted results, and the results are similar ( See figure 2 ), compared with NCBIBLAST after sequencing, the nucleotide sequence has a homology of 83% with E-33. According to the typing standard, it can be judged that the virus is E-33, and part of the VP1 in the genome of the virus can be obtained the nucleotide sequence of the gene;

[0063] (2) According to the conserved type of the 5' end and 3' end of the enterovirus genome, and the nucleotide sequence of part of the VP1 gene obtained in step (1), design primers; in two sections, design primers: EV1F–E332R (approx. 2400bp), E333F-EV8R (about 5000bp) (see Table 6), using viral RNA as a template, EV1F-E332R (about 2400bp), E333F-EV...

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Abstract

The invention discloses an amplification method for a whole genome aiming at different enterovirus serotypes. The method comprises the following steps: extracting RNA of the viruses, carrying out RT-PCR, sequencing and comparison on the VP1 area of the RNA of the viruses by adopting the universal primers 222-224 of the enteroviruses, and determining the serotypes of virus strains by utilizing a molecular biology experimental technology; and designing corresponding primers, namely, designing the primers by utilizing the conservative property of the enteroviruses 5'UTR and 3'UTR, and carrying out corresponding RT-PCR amplification, sequencing and comparison according to the above paired primers till the nucleotide sequence of the whole genome is obtained. The method provided by the invention has the advantages of high amplification speed, as well as simplicity, convenience and feasibility, the cost can be greatly reduced, and the operation is efficient and rapid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for amplifying whole genomes of different enterovirus serotypes. Background technique [0002] Human enterovirus (HEV) belongs to the genus Enterovirus of the family Picornaviridae. According to the current taxonomy, enteroviruses are mainly divided into four groups: human enteroviruses A, B, C and D, with a total of more than 100 species. Serotype, group A includes CV-A2~8, CV-A10, CV-A12, CV-A14, CV-A16, EV-A71, CV-A76 in Coxsackievirus A (CV-A) , CV-A89-CV-A92 and CV-A119; Group B includes all ECHO viruses (echoviruses, E-) and Coxsackie virus group B (CV-B), as well as CV-A9 and EV-B69, EV- B73-B75, EV-B77-88, EV-B93, EV-B97-98, EV-B100-101, EV-B106-107 and EV-B110; Group C includes CV-A1, CV-A11, CV-A13 ,CV-A17,CV-A19-CV-A22,CV-A24,EV-C95,EV-C96,EV-C99,EV-C102,EV-C104,EV-C105,EV-C109,EV-C116-EV -C118, Polio1-3; Group D includes EV-D68, EV-D70, EV-D94, EV-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701C12Q1/6806C12Q1/6869
Inventor 马绍辉张婕刘建生张海浩
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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