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袋鼠疱疹病毒1型实时荧光PCR方法的建立

点击数:4275  时间:2019-03-01 09:00:40  来源: 中国动物检疫      作者: 王建华等

为实现袋鼠临床样本中疱疹病毒1型(Macropodid herpesvirus 1MaHV-1)的出入境快速检测,基于MaHV-1gB基因序列设计特异引物和TaqMan探针,建立了一种MaHV-1荧光PCR检测方法。试验结果显示,该方法只对MaHV-1 gB基因呈现特异性扩增,与禽传染性喉气管炎病毒、伪狂犬病毒和牛传染性鼻气管炎病毒不发生交叉反应,对阳性标准质粒对照(pCR-MaHV-1-gB)的最低检测限为8个拷贝数/反应。该方法的组内和组间试验的Ct值变异系数介于0.17%~0.96%之间,具有良好的重现性。试验结果表明,本研究建立的实时荧光PCR方法可用于袋鼠MaHV-1的病原学检测。


Development of a Real-time PCR for Detection ofMacropodid Herpesvirus 1

For rapidly detecting macropodid herpesvirus 1MaHV-1in clinic samples from kangaroos at entry-exit portsa real-time PCR was developed by designingprimers and probes based on the sequence of gB gene. The results showed themethod could only amplify the MaHV-1 gB gene and no cross-reaction with otherkinds of virus was observedincluding infectious bovine bronchitis virusavian laryngotracheitis virus andpseudorabies virus. The detection limit was 8 copies/reaction for the positivestandard plasmid controlpCR-MaHV-1-gB. The coefficients of variationCVof intra-assay and inter-assay were between 0.17 % to 0.96 %showing good repeatability. In conclusionthe established method was applicable forpathogenic detection of MaHV-1 from the kangaroos.

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