感染细胞与痘疹样本中山羊痘病毒PCR检测方法的建立

比较Trizol法和SDS-蛋白酶K法提取山羊痘病毒DNA后,根据山羊痘病毒P32基因设计引物,建立了能检测感染细胞培养物与组织病料的PCR方法,结果显示以SDS-蛋白酶K法提取的病毒DNA在含量与纯度上均明显高于Trizol法;该PCR方法对山羊痘标准毒株细胞感染物能扩增出特异性的DNA条带,最小检出量为40.625ng;且能检出山羊痘疫苗毒Y株、贵州现场分离毒LD株和QL株的细胞感染物以及山羊痘疹样本中病毒DNA,而对鸡痘疫苗毒株感染细胞、正常细胞及正常山羊皮肤均为阴性反应。这些结果表明,建立的PCR方法具有特异性强、可靠性好、灵敏度高等特点,可用于山羊痘的临床诊断。

Establishment of PCR for detection of goat poxvirus in affected cells and pock samples

After two methods of Trizol methods and SDS-proteinase K were compared for extraction of goat poxvirus(GPV) DNA,the primers sequences to detect GPV in affected cells and pock samples were designed according to the P32 gene sequences published in the GenBank database.The results showed that the content and purity of viral DNA extracted by SDS-proteinase K were obviously better than that by Trizol method.And a specific DNA fragment 963 bp was amplified from DNA extracts of GPV referent strain B in infected cells of with the minimum detection 40.625 ng,and this 963 bp fragment is displayed in DNA samples extracted from infected cells of GPV vaccine strain Y,isolation strain LD and QL and pock samples,but not in DNA samples of infected cells of fowlpox vaccine strain,normal cells and healthy goat skins.These results suggested that this PCR is a specific,reproductive and sensitive method which can be used for clinical diagnosis and detection of GPV infection.