绵羊肺腺瘤LAMP快速诊断方法的初步建立(英文)

THE PRIMARY ESTABLISHMENT OF RAPID LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR DETECTION OF EXOGENOUS JAAGSIEKTE RETROVIRUS IN SHEEP

为了建立一种便捷、灵敏的绵羊肺腺瘤病毒(JSRV)检测技术,本研究根据exJSRV LTR设计2对特异性引物,建立JSRV的LAMP(loop-mediated isothermal amplification,环介导等温扩增)检测方法,对反应体系中的MgSO4Betaine Bst DNA PoLymerase dNTP引物浓度等分别进行了优化,并进行敏感性和特异性试验。结果表明:所建立的反应体系在恒温水浴锅中作用60min即可得到其特有的阶梯状条带,而且对内源性绵羊肺腺瘤病毒(enJSRV)的扩增结果为阴性,该方法对JSRV前病毒DNA的最小检测限为10拷贝,灵敏性高于一步PCR方法。LAMP和特异性PCR及套式PCR方法检测临床样品的符合率分别为92%和100%。该方法为绵羊肺腺瘤病的临床检测和基层检疫提供了一种简便、实用的方法,可望在绵羊肺腺瘤病的流行病学调查及防控方面起重要作用。基于本技术的专利已获国家知识产权局批准(专利号ZL 2012 1 0274280.7)。

The jaagsiekte retrovirus（JSRV） is the pathogen that causes ovine pulmonary adenocarcinoma（OPA）,which is one of the major infectious viral diseases in sheep.In this study,a method of loop-mediated isothermal amplification was developed and evaluated to detect JSRV infections.A set of four specific primers was designed to recognize four distinct genomic sequences in the long terminal repeat（LTR） of the JSRV.A LAMP assay specifically detected exogenous JSRV in the infected tissues of OPA sheep in reactions that were carried out for 60 minutes in a thermostatic water bath and detected a minimum of 10 copies of the JSRV LTR/reaction in a testing plasmid.The sensitivity of this method is comparable to that of nest-PCR and is 10 times higher than that of standard PCR.Tissue samples from clinically diagnosed and experimentally infected OPA sheep and frozen stored OPA organs were simultaneously examined by LAMP,nest-PCR and standard PCR.The coincidence of the results was 100% between LAMP and nest-PCR and 92% between LAMP and standard PCR.Our results show that LAMP assay is a sensitive,specific and rapid method for the detection of JSRV infections.The patent based on this technique has been authorised by State Intellectual Property Office,P.R.of China（Patent number ZL 2012 1 0274280.7）.