摘要
从广东省1例慢性丙型肝炎病人血清中提取HCVRNA,随机引物逆转录为cDNA后用HCV5’端非编码区(5’NCR)特异引物进行聚合酶链反应(PCR)。扩增产物302bp,经补齐和提纯后插入pUC19质粒,获得的重组质粒pUN采用双脱氧链终止法测定核苷酸序列,与国内外多个株比较,核苷酸同源性介乎92.69%~100%,其中与HCVⅡ(1b)型的同源性最大。本文的测序结果可为引物设计提供依据。获得的HCVcDNA在常规PCR步骤中用于设立有效的模板对照,对消除假阴性及评估试剂有重要意义。
HCV RNA derived from the serum of a patient with chronic hepatitis C was extracted before it was converted to cDNA by reverse transcription using random primers. Polymerase chain reaction (PCR) was performed with primers specific for HCV 5'NCR. Amplified product with 302bp was subsequently filled in 3' recessed ends, isolated, purified and inserted into pUC19 plasmid vector. The recombinant plasmid pUN obtained was sequenced by dideoxy nucleotide chain termination method. A comparison of the nucleotide sequence of HCV 5'NCR with several previously reported strains showed the homology to be 92.69% ̄100% and most identical to strains belong to genotype Ⅱ. The sequence of the HCV 5'NCR determined in this report was useful for the selection of primers. The cDNA clone constructed can be used as effective positive control template for PCR, which will be helpful in eliminating false negative results and evaluating the quality of reagents.
出处
《中山医科大学学报》
CSCD
1997年第1期5-8,共4页
Academic Journal of Sun Yat-sen University of Medical Sciences
基金
广东省科委基金