畜牧与兽医2011，Vol.43Issue(7)：6-11,6.
绵羊肺腺瘤病毒实时荧光定量PCR检测方法的建立
Establishment of a Real-time qPCR for detection of jaagsiekte sheep retrovirus
摘要
Abstract
In order to establish a Real-time qPCR for detection of Jaagsiekte sheep retrovirus (JSRV) , the conservative fragment of era; gene was amplified from genomic DNA isolated from lung tumour tissues by PCR with specific primers and TaqMan probe was designed according to the sequences from JSRV-NM strain. The PCR product was cloned into pGEM-T and the recombinant plasmid was used as a standard quantitative template to develop the Real-time qPCR. The standard curve was Y= -3. 308A"+47. 848, and the correlation coefficient was 0. 991. The Real-time qPCR method had good reproducibility, and was more sensitive than standard PCR. Blood and tissue samples from the different groups of sheep were tested for detection of JSRV proviral DNA by Real-time qPCR. The results showed that the peripheral blood leukocytes, lungs and mediastinal lymph nodes, and nostrils fluid in both B and C groups were positive, and the proviral DNA loads were higher in the lungs than those in peripheral blood leukocytes. In all sheep from group D, the proviral DNA could be detected in the mediastinal lymph nodes, but the sheep had no clinical signs of Sheep pulmonary adenomatosis (SPA). One sheep from group E was positive in lung, and group A was negative. This study would be useful in investigating the JSRV infection at flock level.关键词
绵羊肺腺瘤病毒/实时荧光定量PCR/TaqMan探针/前病毒DNAKey words
jaagsiekte sheep retrovirus/ Real-time qPCR/ proviral DNA分类
农业科技引用本文复制引用
斯日古楞,么宏强,马学恩,王升启..绵羊肺腺瘤病毒实时荧光定量PCR检测方法的建立[J].畜牧与兽医,2011,43(7):6-11,6.基金项目
国家自然科学基金(30260083,31060332) （30260083,31060332）
教育部高等学校博士点专项研究基金(20040129001). （20040129001）
