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犬细小病毒2c亚型毒株的分离与鉴定
毛娅卿
0
(中国兽医药品监察所)
摘要:
采集一疑似犬细小病毒(Canineparvo-virus,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上已经公布的细小病毒的全基因组序列以及相关的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1755bp。进化分析显示,该毒株属于CPV-2c型,并命名为CPV-2c-Guangxi23。
关键词:  犬细小病毒  分离与鉴定  免疫过氧化物酶单层细胞染色法  
DOI:
投稿时间:2019-04-26修订日期:2019-08-09
基金项目:
Isolation and Identification of Canine Parvovirus 2C Subtype
(China institute of Veterinary Drug Control)
Abstract:
The faeces samples were collected from a dog suspected infected by canine parvovirus (CPV),CPV in the samples was 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).Samples that cultured with or without serum were titrated by IPMA.The VP2 gene of the virus was amplified by PCR and sequenced by DNA MAN software. Compared with the whole genome sequence of parvovirus and the nucleic acid sequence of related proteins published on NCBI, the virus strain was identified and the amino acid sequence was deduced. The results showed that the virus was positive for CPV nucleic acid. After 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-2c type and was named CPV-2c-Guangxi23.
Key words:  Canine parvovirus  Isolation  Identification  Immunoperoxidase monolayer assay  

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