应用多重PCR检测和区分3个型的马疱疹病毒

针对马疱疹病毒(EHV)的EHV-1、EHV-2和EHV-4糖蛋白B基因序列,设计、合成了3对特异性引物进行多重PCR,不仅可以在数小时内分别检测这3个型的EHV,而且在同一反应系统内可以清晰地区分EHV-1、EHV-2和EHV-4,其PCR产物大小分别为226、3335、70bp,符合预期的片段大小,序列分析证实与已发表的序列一致;该检测方法的灵敏度达到103TCID50;分别从血清学阳性但病毒分离为阴性的1匹进口马组织样品和一些出口前检疫马的鼻咽样品检测到EHV-1和EHV-4特异性核酸。

Detection and differentiation of three types of equine herpesviruses by multiplex PCR

A multiplex PCR assay was developed to detect and distinguish equine herpesviruses EHV-1, EHV-2 and EHV-4 in the same reaction within several hours, using 3 pairs of specific primers for gB gene of three different types of equine herpesviruses. The predicted PCR products of EHV-1, EHV-2 and EHV-4, which were 226, 333 and 570bp, respectively, could be readily separated by gel electrophoresis. The specificity of these amplifications was confirmed by determining their nucleotide sequences of the PCR products, which agreed with the published sequence of these gB genes, and its sensitivity was 10~(3) TCID_(50). Tissues from one seropositive horse of post-arrival quarantine was positive for EHV1 by PCR but negative by virus isolation, whereas some nasopharyngeal swAbs from seropositive horses of pre-export quarantine were positive for EHV4 by PCR but negative by virus isolation. The results showed that the quick, specific, sensitive and reliable multiplex PCR assay was a practical alternative to identify low level of (non-)(infectious) equine herpesviruses or presence of latent forms in horse tissue.