伪狂犬病毒实时荧光定量PCR方法的建立

摘要

Real-timefl uorescence quantitative PCR (qPCR) assay was developed using a pair of primers and aTaqMan probe that were designed according to the sequences of thegB gene of Pseudorabies virus (PRV). This qPCR assay allowed for rapid, sensitive and specifi c detection of PRV DNA. The detection limit was as low as 10 copies/μL viral DNA, which was 100 times more sensitive than the conventional PCR. There was no cross-reactivity with other related viruses, such as Classical swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus type 2 (PCV2), indicating that the assay was highly specifi c to PRV. The distributions with a coeffi cient of variation to be less than 1% showed that the assay had good reproducibility. Therefore, the qPCR assay was a valuable tool for rapid detection and quantifi cation of PRV.%根据伪狂犬病毒(Pseudorabies virus,RV)gB基因保守序列,设计合成一套特异引物和TaqMan探针,建立了一种快速、敏感和特异的检测伪狂犬病毒的实时荧光定量PCR方法.该方法可检测到相当于10 copies/μL的病毒DNA,比常规PCR检测方法高约100倍,敏感性较强;该方法检验猪瘟病毒、猪繁殖与呼吸综合征病毒和猪圆环病毒2型呈现阴性结果,特异性强;变异系数小于1%,具有良好的重复性.本研究建立的实时荧光定量PCR方法为临床上对伪狂犬病毒的检测和定量奠定了坚实基础.