RT-PCR-RFLP方法鉴别基因Ⅰ型和基因Ⅲ型日本脑炎病毒

doi:10.11843/j.issn.0366-6964.2014.09.025

Abstract

Abstract:

RT-PCR-RFLP was established to detect and differentiate Japanese encephalitis virus genotype Ⅰ (GⅠ) and genotype Ⅲ (GⅢ).A pair of primers was designed according to the sequences of Japanese encephalitis virus to amplify the C-PrM-E gene.One restriction enzyme (SpeⅠ) was selected to generate the polymorphisms in different restriction fragments length for genotype discrimination.Seventy-eight clinical samples were detected by RT-PCR-RFLP for detecting and genotyping.The results showed that a 819 bp target fragment was amplified by RT-PCR from genomic RNA of JEV GⅠ strains which could be digested with SpeⅠ into 2 fragments of 568 and 251 bp when analyzed with RFLP，the GⅢ strains could be digested into 3 fragments of 446，251 and 122 bp，while the mixture of GⅠ and GⅢ strains could be digested into 4 fragments of 568，446，251 and 122 bp，respectively，but not amplified from swine fever virus，porcine parvovirus，porcine reproductive and respiratory syndrome virus，porcine circovirus and pseudorabies virus.The lowest concentration of RNA could be detected was 1.5 pg•μL^-1.Seventy-eight samples were tested by RT-PCR-RFLP，and the RT-PCR-RFLP results showed that 6 of these were GⅠ and 8 of these were GⅢ，and those results by RT-PCR-RFLP correlate well to those determined by sequencing.The RT-PCR-RFLP technology is a useful tool for detecting and rapid genotyping of JEV GⅠ and GⅢ.

CLC Number:

S852.659.6

ZHANG Liang，TIAN Geng,SHI Shuang-yan，YUAN Lei，LIU Han-yang，HUANG Xiao-bo，WU Rui，WEN Xin-tian，WEN Yi-ping，CAO San-jie. Development of a RT-PCR-RFLP Assay for Differentiation of Japanese Encephalitis Virus Genotype Ⅰ and Genotype Ⅲ[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(9): 1555-1560.

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Development of a RT-PCR-RFLP Assay for Differentiation of Japanese Encephalitis Virus Genotype Ⅰ and Genotype Ⅲ

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