多重PCR检测HBV和HCV方法的建立

Establishment of Multi-primer PCR Identifying HBV and HCV

目的:建立可同时检测血清HBVDNA和HCVRNA的多重PCR。方法:利用已知的基因序列设计了对于不同亚型HBV保守的C基因区的一对引物HBV-P623及对于不同亚型HCV高度保守的位于5’端非编码区(5’-NCR)的两对引物HCV-P289和HCV-P174,扩增经基因释放剂(Genereleaser)处理的HBV和HCV阳性血清。结果:多重引物、逆转录套式PCR反应体系可同时扩增HBV DNA和HCV RNA,分别在623bp和174bp处出现特异DNA扩增带。该反应体系具有较高的特异和敏感性,受检血清中仅含有10fgHBV DNA和5CID HCV RNA即可检出。血清标本不需要提取核酸,可直接进行PCR。用此体系检测30例输血后病人血清,其中15例为HBV感染,8例为HCV感染,7例为阴性。结论:多重PCR检测HBV及HCV的方法具有简便性和实用性。

Objective To establish multi-primer FCR identifying HBV and HCV simultaneously. Methods A pair of primer in conservative C gene of different types of HBV and two pairs of primer in highly conservative 5' non-coding regions (5' -NCR) of different types of HCV were synthesized and the positive serums of HBV and HCV treated by the gene rdeaser were amplified by multi-primer PCR. Resuls HBV DNA and HCV RNA can be specifically amplified simultaneously in one system by multi-primer nested PCR. The zone of DNA amplification appeared in 623bp and 174bp respectively. The serums from 30 patients with hepatitis, of whom, 15 were infected with HBV, 8 with HCV and 7 had negetive serum, were detected by the system. Gndusions The system was specific and sensitive in detecting HBV DNA and HCV RNA, and can identify 10fg HBV DNA and 5 Chimpanzee infection dose (CID) HCV RNA. The serum can be directly used as model for PCR without extraction of DNA and HCV.