犬细小病毒2c亚型毒株的分离与鉴定

Isolation and Identification of Canine Parvovirus 2C Subtype

采集一疑似犬细小病毒(CPV)患犬的粪便,用纳米PCR方法对病料进行CPV检测,并应用猫肾细胞F81进行病毒分离培养,采用免疫过氧化物酶单层细胞染色法(IPMA)检测病毒在F81细胞中的增殖动态。利用PCR扩增分离病毒的VP2基因,测序结果用DNAMAN软件对获得的VP2基因序列进行拼接,并与NCBI上已经公布的犬细小病毒的全基因组序列以及相关蛋白的核酸序列进行比较,确定其所分离的病毒株型并推导氨基酸序列。结果表明,纳米PCR检测结果为CPV核酸阳性,病料接种到F81细胞培养后出现明显细胞病变(CPE),血清学鉴定为CPV抗原阳性,序列测定分析表明,该毒株VP2基因开放阅读框(ORF)为1755 bp。进化分析显示,该毒株属于CPV-2c型,并命名为CPV-2c-Guangxi23。

The faeces samples which were collected from a dog suspected infected by canine parvovirus(CPV)were detected by nano polymerase chain reaction(PCR).Then the samples were innoculated into F81 cells to isolate CPV.The proliferation of the virus in F81 cells was detected by immunoperoxidase monolayer cell staining(IPMA).The VP2 gene of the virus was amplified by PCR and sequenced by DNAMAN software.Compared with the whole genome sequence and the nucleic acid sequence of related proteins of parvovirus published on NCBI,the strain of the virus was identified and the amino acid sequence of the strain was deduced.The results showed that the virus was positive for CPV nucleic acid.After the virus was inoculated into F81 cells,obvious cytopathic changes(CPE)were observed.Serological identification showed that the virus was positive for CPV antigen.Sequence analysis showed that the open reading frame(ORF)of VP2 gene of the strain was 1755 bp.Evolutionary analysis showed that the strain belonged to CPV-2 c type and was named CPV-2 c-Guangxi23.