绵羊肺腺瘤病毒巢式RT-PCR检测方法的建立与验证  

Establishment and verification of a nested RT-PCR method for detection of jaagsiekte sheep retrovirus

作　　者：郭文庆[1] 孙晓琳[1] 冷冉 刘淑英[1] 

机构地区：[1]内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特010018

出　　处：《中国兽医科学》2017年第1期23-30,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31360597);内蒙古科技厅应用研究项目(201502070);内蒙古草原英才创新团队项目(20151031)

摘　　要：根据Gen Bank上发表的外源性绵羊肺腺瘤病毒(exJSRV)的基因序列,分别设计合成巢式RT-PCR引物及扩增exJSRV-env全基因的引物,建立exJSRV的检测方法。将病肺的巢式外套与内套RT-PCR扩增产物与载体连接,对重组质粒送检测序进行序列分析。结果显示,巢式外套与内套RT-PCR扩增产物的序列与JQ837489.1序列的同源性分别为99.7%和99.4%。通过对3份鼻液以及75份血样的临床检测,将检测阳性的1份鼻液和2份血样送检测序,序列比对结果显示,鼻液、血样的巢式外套和内套RT-PCR扩增产物与JQ837489.1的同源性分别为99.8%、99.7%、99.7%和98.9%、100%、99.4%,且扩增基因序列的氨基酸序列中具有exJSRV特有的致病性YXXM序列。敏感性试验表明,巢式RT-PCR诊断方法的敏感性是普通RT-PCR的10倍,最低能够检测出9.72 fg的标准模板RNA。说明本试验所建立的方法,具有良好的临床应用价值,灵敏性高、特异性好,对exJSRV的临床早期检测提供了一种方便、可靠、经济实用的诊断方法。Based on the genome sequence of exogenous jaagsiekte sheep retrovirus（exJSRV） available in GenBank,the nested RT-PCR primers and complete sequence of exJSRVprimers were designed and synthe- sized,and the nested RT-PCR detection method for exJSRVwas established. The nested outer and inner RT-PCR amplified product of ovine pulmonary adenomatosis lung were linked to a carrier,and then sequenced. The results showed that the sequences of the nested outer and inner RT-PCRamplified produc- tions were 99.7% and 99.4% homologous with JQ837489.1,respectively. Based on clinical tests of 3 nasal discharge samples and 75 blood samples,the samples（1 nasal discharge sample and 2 blood sam- ples） that were tested positively were sequenced,and sequence alignment showed that the sequences of the nested outer and inner RT-PCRamplified product of the nasal discharge samples and the blood sam- ples were 98.8% ,99.7% ,99.7% ;98.9% ,100% ,99.4% homologous with JQ837489. l,respectively. The amino acid sequence of the amplified gene contained pathogenic sequence YXXMpeculiar to exJSRV. The sensitivity test proved that the sensitivity value of the nested RT-PCR diagnostic method was i0 times higher than that of the ordinary RT-PCR method,and the lowest sensitivity value of the nested RT-PCR was enough to detect 9. 72 fg of the standard template P^A. It indicates that the setup of the method in this test has a high clinical practice value with a higher sensitivity and a better specificity,which provides a convenient,reliable,economical and practical diagnosis method for early clinical detec- tion of sheep pulmonary adenomatosis.

关 键 词：绵羊肺腺瘤病 巢式PCR 外源性绵羊肺腺瘤病毒 内源性绵羊肺腺瘤病毒 

分 类 号：S852.65[农业科学—基础兽医学]