绵羊肺腺瘤病特异性PCR及套式PCR诊断方法的建立  

A Specific PCR Assay and a Nested PCR Assay for the Screening of Ovine Pulmonary Adenomatosis

作　　者：罗军荣[1,2] 刘霄卉[1] 余群英[2,3] 张淑琴[1] 周建华[4] 马学恩[1] 

机构地区：[1]内蒙古农业大学动物科学与医学学院,呼和浩特010018 [2]江西农业大学动科院,南昌330045 [3]江西省铅山县农业局,铅山334500 [4]中国农业科学院哈尔滨兽医研究所,哈尔滨150001

出　　处：《病毒学报》2009年第2期125-130,共6页Chinese Journal of Virology

基　　金：国家自然科学基金项目(30660136);教育部高等学校博士学科点专项科研基金项(20040129001);兽医生物技术国家重点实验室开放课题基金(NKLVB200801)

摘　　要：绵羊肺腺瘤是由外源性反转录病毒JSRV引起的接触性传染的肺肿瘤性疾病。感染羊没有针对JSRV的循环抗体,但在感染羊的肺肿瘤组织、淋巴网状系统及外周血单核细胞中可检测到外源性前病毒exJSRV及JSRV转录产物。健康羊基因组中存在15~20拷贝的内源性前病毒enJSRV,内外源性前病毒的结构基因高度相似,而在U3区存在差别,因而,设计了针对exJSRVU3区的特异性引物并在国内首次建立了一步法特异性PCR检测法及套式PCR检测法。以700ng健康羊基因组DNA为背景,梯度稀释阳性质粒pJSRV-LTR作为模板,比较两种方法的敏感性,结果表明套式法的敏感性是一步法的10倍以上,套式法也是目前可用于检测感染羊血液样品的唯一方法,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。Ovine pulmonary adenomatosis （OPA） is a naturally occurring contagious lung tumor of sheep which was caused by an exogenous retrovirus of sheep, jaagsiekte retrovirus （JSRV）. Although no specific circulating antibodies against the virus coud be detected in infected sheep, exogenous JSRV proviral DNA sequences （exJSRV） and JSRV RNA transcripts could be detected in lung tumors, lyrnphoreticular system and peripheral blood mononuclear cells （PBMC） from sheep affected by OPA. The sheep genome carried 15 to 20 copies of endogenous retrovirus loci （enJSRV） that were similar to JSRV in structural genes but the divergene in U3. Therefore, primers specific for the U3 sequences of exJSRV were designed for the specific PCR and nested PCR （n-PCR）. Sensitivity between specific PCR assay and n-PCR assay was compared by using serial dilutions of positive plasmid pJSRV-LTR in a background of 700ng sheep genome DNA. Sensitivity of n-PCR was ten-fold higher than specific PCR. The n-PCR was only available in blood test for detection of JSRV infected sheep and might be useful in epidemiological studies and disease control of OPA.

关 键 词：绵羊肺腺瘤 绵羊肺腺瘤病毒 特异性PCR 套式PCR 

分 类 号：S852.65[农业科学—基础兽医学]