Tiwari A K, Kataria R S, Desai G, Butchaiah G, Bandyopadhyay S K
National Biotechnology Centre, Indian Veterinary Research Institute, Izatnagar, UP.
Vet Res Commun. 2000 Sep;24(6):401-9. doi: 10.1023/a:1006426301134.
The reverse transcription-polymerase chain reaction (RT-PCR) was standardized to amplify the VP-7 gene sequences of an Indian isolate of bluetongue virus serotype 23. Using two different sets of primers, a sequence of 1156 bp comprising the complete coding sequence of the VP-7 gene and its 770 bp internal sequence were amplified. The sensitivity of RT-PCR, using these two sets of primers individually was 40 pg and 4 pg, with the external and internal primers, respectively, whereas the nested PCR was 100-fold more sensitive than the single PCR with the external primers. Further, by restriction enzyme digestion of the 1156 bp amplicon, using CfoI, PstI and TaqI enzymes, the Indian isolate was found to be genetically different from isolates from the United States and Australia. RT-PCR and restriction enzyme digestion were applied to detect virus directly in blood samples taken from sheep suspected of bluetongue virus infection.
逆转录聚合酶链反应（RT-PCR）被标准化用于扩增蓝舌病毒血清型23印度分离株的VP-7基因序列。使用两组不同的引物，分别扩增出包含VP-7基因完整编码序列的1156 bp序列及其770 bp内部序列。单独使用这两组引物时，RT-PCR的灵敏度分别为40 pg（外部引物）和4 pg（内部引物），而巢式PCR比使用外部引物的单重PCR灵敏度高100倍。此外，通过使用CfoI、PstI和TaqI酶对1156 bp扩增子进行限制性酶切，发现印度分离株在基因上与来自美国和澳大利亚的分离株不同。RT-PCR和限制性酶切被用于直接检测从疑似感染蓝舌病毒的绵羊采集的血液样本中的病毒。
