基因2型牛病毒性腹泻病毒RT-PCR检测方法的建立和初步应用

Establishment of RT-PCR assay for detection of bovine viral diarrhea virus genotype 2

根据已发表的牛病毒性腹泻病毒基因1型(BVDV-1)和2型(BVDV-2)5'非编码区(5'-UTR)的序列,分别设计了针对BVDV-1型、BVDV-2型以及针对BVDV-1和BVDV-2两型的特异性引物和通用引物,以标准参考株BVDV-1NADL株和BVDV-2890株为对照,建立了分别能检测BVDV-1、BVDV-2和BVDV-1/BVDV-2的RT-PCR检测方法。在此基础上,进一步对疑似BVDV-2感染牛的临床病料组织(脾、淋巴结、心肌、血液等)检测结果表明,在所检测的18份样品中,BVDV-2阳性8份(44%)。经RT-PCR鉴定为阳性的组织病料,进一步通过MDBK细胞进行病毒分离,病毒分离率为100%;结合感染细胞病变观察,间接免疫荧光试验RT-PCR和序列测定鉴定,所分离的病毒均为BVDV-2。上述研究表明,该RT-PCR检测方法敏感、特异;并证实我国牛群已存在BVDV-2的污染或感染。

A RT-PCR assay was developed using primers specific to Bovine Viral Diarrhea Virus genotype 1 （BVDV-1）, genotype 2 （BVDV-2）, or both genotypes. The assay was able to differentiate reference strains NADL （BVDV-1） and 890 （BVDV-2）. Test on clinical samples from a suspicious BVDV-2 infection cattle showed that 44% tissue samples, including spleens, lymphonodes, hearts and blood were BVDV-2 positive. The BVDV-2 virus could be isolated from all RT-PCR positive samples on MDBK cells with typical CPE, which was confirmed by indirect immunofluorescence assay, RT-PCR and sequence data. This is the first isolation of BVDV type 2 in China.