用多重及套式PCR同时检测血清中的HBV和HCV方法的建立

MULTANEOUS AMPLIFICATION OF HBV AND HCV IN SERUM USING MULTIPLEX AND NESTED PCR

首次应用多重及套式ＰＣＲ技术同时检测人血清中的ＨＢＶ和ＨＣＶ。将抽提的ＨＢＶＤＮＡ和（或）ＨＣＶＲＮＡ在含ＡＭＶ逆转录酶、ＴａｑＤＮＡ多聚酶以及ＨＢＶ和ＨＣＶ外套引物的ＰＣＲ缓冲液中进行逆转录后连续进行ＰＣＲ扩增，以第一轮扩增产物为模板，在含ＨＢＶ和ＨＣＶ内套引物的ＰＣＲ反应体系中进行第二轮扩增，产物经电泳后，以ＤＮＡ分子量标志物或巳知片段做参考，出现５２３ｂｐ和（或）２６０ｂｐ产物条带分别为ＨＢＶ和（或）ＨＣＶ单项或重叠感染。因选择高度保守的ＨＢＶ和ＨＣＶ引物，并进行了包括将逆转录与第一轮ＰＣＲ结合进行等多项改进，故本方法具有特异、敏感、通用、简便、快速和经济等优点，减少了交叉污染的机会，有很高的临床实用价值，亦为献血员筛选提供一种可靠的方法。

A method of simuItaneous detection of HBV and HCV in serum using multiplex and nested PCR in the same reaction system is reported. The extracted HBV DNA and/or HCV RNA are reverse transcribed, and then amplified in PCR buffer containing AMV reverse transcriptase, Taq DNA poly merase, HBV and HCV outer primers; the second round muItiplex PCR is done by using the first round PCR products as ternplates in the system composing of HBV and HCV inner primers. By referring the DNA marker or known size fragments, infection or coinfection of HBV and/or HCV are determined if the 523 bp and/or 260 bp bands are seen via electrophoresis. Because the primers are deduced from the high conservative HBV DNAs gene and HCV RNA 5’ uncoding region, and many steps, such as the combina tion of reverse transcription with first round PCR amplification, are improved, this method is highly spe- cific, sensitive, universal, rapid, simple as well as economic, and reduces cross contamination, It pro- vides potential value of clinical diagnostic use and a reliable choice of screening blood donor.