RT-PCR和酶切方法区分猪瘟疫苗毒与野毒的研究  

Differentiation Between Vaccine Strain and Field Isolates of Classical Swine Fever Virus Using Polymerase Chain Reaction and Restriction Test

作　　者：赵耘[1] 秦玉明[1] 张广川[1] 赵启祖[1] 宁宜宝[1] 

机构地区：[1]中国兽医药品监察所,北京100081

出　　处：《微生物学通报》2006年第3期82-87,共6页Microbiology China

基　　金：国家重点基础研究发展规划项目(No.G1999011905)

摘　　要：建立了套式RT-PCR与限制性内切酶酶切相结合的区别猪瘟兔化弱毒疫苗株与猪瘟病毒田间分离株的诊断方法。通过对猪瘟兔化弱毒疫苗株与猪瘟石门株E2基因主要抗原编码区序列进行限制性内切酶酶切位点分析,分别找出猪瘟兔化弱毒疫苗株与猪瘟石门株各自独有的限制性内切酶酶切位点,结果二者分别有10和16个独有的限制性内切酶的酶切位点;分别对17株猪瘟病毒E2基因主要抗原编码区序列进行这26个限制性内切酶酶切位点分析,结果表明有3个限制性内切酶(HgaI、Hin8I及Hsp92I)的酶切位点在HCLV株序列是独有的;利用HgaI限制性内切酶分别对HCLV、HCVSM及5株不同基因群的猪瘟病毒田间分离株进行酶切鉴定,结果只有HCLV株能够被HgaI酶切成2个片段,而其它的毒株则不能被切开。同时测定了套式RT—PCR方法的敏感性及特异性,结果其敏感性可达到0．2MLD,而对BDV以及BVDV均不能特异扩增。本方法的建立无疑对猪瘟在我国的控制和消灭具有重要的意义。A nest RT-PCR/restriction test has been developed in order to distinguish the lapinised vaccine strain from field isolates of classical swine fever virus. The rostriction enzyme cut sites mapping of the major coding sequence of E2 gene lapinised vaccine strain and ShiMen strain of classical swine fever virus have been compared. Ten and sixteen unique restriction markers have been found in the lapinised vaccine strain and ShiMen strain. The restriction enzyme cut sites mapping of the twenty six unique restriction marker in the major coding sequence of E2 gene of 17 classical swine fever field isolates have been analyzed. Only 3 sites （ HgaI 、 Hin8I 、 Hsp92I） are present in the lapinised vaccine strain sequence. Two paris of nested primers and a criteria of analysis have been designed for HgaI restriction marker site. The tests have been conducted first on the lapinised vaccine strain and ShiMen strain of classical swine fever virus resulting in predicted restrection patterns. Finally, the tests have been applied to 5 field isolates of different gene group analyzed by phylogenetic study, The result showed that only HCLV strain gene can he cut to 2 fragment by HgaI , and ShiMen strain and 5 field isolates cant be cut. At the same time the sensitivity and specificity of nest RT-PCR have been tested. The sensitivity is 0. 2MLD. The specific fragment of BDV and BVDV were not obtained by the nest RT-PCR. These results showed that the development of the nest RT-PCR/restriction tests is very important for the control and perish of classical swine fever in china.

关 键 词：猪瘟兔化弱毒 猪瘟田间分离毒 RT-PCR 酶切 

分 类 号：S852.65[农业科学—基础兽医学]