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

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (9): 1555-1560.doi: 10.11843/j.issn.0366-6964.2014.09.025

RT-PCR-RFLP方法鉴别基因Ⅰ型和基因Ⅲ型日本脑炎病毒

张亮^1,2，田耕^1,2，石双艳^1,2，袁磊^1,2，刘澣扬^1,2，黄小波^1,2,3，伍锐^1,2,3，文心田^1,2,3，文翼平^1,2,3，曹三杰^1,2,3*

（1．四川农业大学预防兽医研究所猪病研究中心，雅安 625014；2．四川农业大学动物医学院人兽共患病研究室，成都 611130；3．农业部兽用药物与兽医生物技术四川科学观测实验站，雅安 625014）

收稿日期:2014-01-25 出版日期:2014-09-23 发布日期:2014-09-23
通讯作者: 曹三杰，教授，博士，博士生导师，E-mail：csanjie@gmail.com
作者简介:张亮（1990-），男，重庆开县人，硕士，主要从事动物传染病研究，E-mail：zhangliang215@163.com
基金资助:
四川省科技支撑计划（2013SZ0068）

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

ZHANG Liang^1,2，TIAN Geng^1,2,SHI Shuang-yan^1,2，YUAN Lei^1,2，LIU Han-yang^1,2，HUANG Xiao-bo^1,2,3，WU Rui^1,2,3，WEN Xin-tian^1,2,3，WEN Yi-ping^1,2,3，CAO San-jie^1,2,3*

(1.Swine Research Center，Institute of Preventive Veterinary Medicine，Sichuan Agricultural University，Ya’an 625014，China；2.Laboratory of Zoonosis，College of Veterinary Medicine，Sichuan Agricultural University，Chengdu 611130，China；3.Sichuan Science-observation Experiment Station of Veterinary Drugs and Veterinary Biological Technology，Ministry of Agriculture，Ya’an 625014，China)

Received:2014-01-25 Online:2014-09-23 Published:2014-09-23

摘要/Abstract

摘要：

建立一种可检测及鉴别基因Ⅰ型和基因Ⅲ型日本脑炎病毒（JEV）的RT-PCR-RFLP方法。通过对JEV基因Ⅰ型（GⅠ）和基因Ⅲ型（GⅢ）核酸序列的比对分析，设计1对特异性引物对JEV C-PrM-E区域部分基因进行RT-PCR扩增，扩增产物经限制性内切酶SpeⅠ消化后于15 g•L^－1琼脂糖凝胶电泳进行RFLP分析，并利用建立的RT-PCR-RFLP方法对78份临床样品进行了JEV的检测及基因型鉴定。结果显示，特异性引物可扩增出长为819 bp的片段，而对猪瘟病毒、猪细小病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒及猪伪狂犬病毒的核酸扩增结果均为阴性。经SpeⅠ酶切，基因Ⅰ型JEV RT-PCR产物被切为568和251 bp 2个条带，基因Ⅲ型JEV RT-PCR产物被切为446、251和122 bp 3个条带，基因Ⅰ型和基因Ⅲ型JEV混合RT-PCR产物被切为568、446、251和122 bp 4个条带，该方法最低可检出1.5 pg•μL－1的JEV RNA。RT-PCR-RFLP结果显示78份临床样品中有14份为JEV阳性，其中6份为基因Ⅰ型，8份为基因Ⅲ型，与核苷酸测序分析结果一致。本研究为日本脑炎病毒的实验室诊断及基因Ⅰ型和基因Ⅲ型的鉴别提供了一种快速有效的分子生物学方法。

关键词: 日本脑炎病毒, 反转录-聚合酶链反应-限制性片段长度多态性, 基因Ⅰ型, 基因Ⅲ型, 鉴别

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.

Key words: Japanese encephalitis virus, RT-PCR-RFLP, genotype Ⅰ, genotype Ⅲ, differentiation

中图分类号:

S852.659.6

张亮，田耕，石双艳，袁磊，刘澣扬，黄小波，伍锐，文心田，文翼平，曹三杰. RT-PCR-RFLP方法鉴别基因Ⅰ型和基因Ⅲ型日本脑炎病毒[J]. 畜牧兽医学报, 2014, 45(9): 1555-1560.

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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RT-PCR-RFLP方法鉴别基因Ⅰ型和基因Ⅲ型日本脑炎病毒

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

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