Lee C W, Hilt D A, Jackwood M W
Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens 30602-4875, USA.
Avian Dis. 2000 Jul-Sep;44(3):650-4.
Diagnosis of the DE072 strain of infectious bronchitis virus (IBV) by the reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) serotype identification test was not possible because the primer used in the RT-PCR did not amplify the S1 gene of the DE072 strain. The 3' end of the polymerase gene and the 5' end of the S2 gene of the DE072 strain were sequenced and compared with the forward and reverse RT-PCR primers, respectively. A 2-bp mismatch at the 3' end of the reverse primer was found. On the basis of these data, a degenerate primer that could amplify the S1 gene of the DE072 strain as well as eight other serotypes of the virus was synthesized. In addition, we were able to differentiate the DE072 strain from all of the other IBV strains examined by RFLP analysis of the RT-PCR product.
由于逆转录聚合酶链反应（RT-PCR）中使用的引物未能扩增出传染性支气管炎病毒（IBV）DE072株的S1基因，因此无法通过RT-PCR和限制性片段长度多态性（RFLP）血清型鉴定试验对该病毒株进行诊断。对DE072株的聚合酶基因3'端和S2基因5'端进行了测序，并分别与RT-PCR正向和反向引物进行了比较。结果发现反向引物3'端存在2个碱基错配。基于这些数据，合成了一种简并引物，该引物能够扩增DE072株以及该病毒其他8种血清型的S1基因。此外，通过对RT-PCR产物进行RFLP分析，我们能够将DE072株与所有其他检测的IBV株区分开来。
