血清乙型肝炎病毒DNA水平对HBV全基因组克隆效率的影响

Impact of HBV DNA level in serum on the cloning efficiency of HBV full length genome

目的:通过比较血清HBV DNA水平对一片段PCR法和两片段PCR法这两种不同方法的HBV全基因组克隆效率的影响,选择出合适的HBV全基因组克隆技术,供后续的HBV的基础和临床研究。方法:采集了慢性乙型肝炎(CHB)患者85份血清,分别用本研究建立的一片段PCR法及两片段PCR法扩增HBV全基因组,经双酶切鉴定后将PCR产物克隆入载体,进行HBV全基因组序列测定。同时用实时荧光定量PCR法检测上述85份血清的HBV DNA滴度。结果:本研究建立的一片段PCR法扩增成功需要的血清HBV DNA浓度高于两片段PCR法,两种方法扩增的效率比较为:一片段PCR法扩增的阳性率低于两片段PCR法(P<0.01)。一片段PCR法的保真性和灵敏度分析发现:保真性:核苷酸的人为突变率为1.13bp/kb;灵敏度:为102个初始模板。浓度大于103的重组质粒DNA80%可以成功扩增,浓度低于该值的扩增效率比较低。结论:血清HBV DNA水平对两种扩增方法的阳性率有一定影响,滴度高低是选择合适PCR扩增方法的依据。HBV DNA滴度大于106copies/L的样本,可选用一片段PCR法的全基因组扩增、克隆技术,而对HBV DNA滴度小于106copies/L样本,则可选用两片段PCR法的全基因组扩增、克隆技术。本研究建立的一片段PCR法扩增HBV全基因组克隆的技术是一种值得推荐的好方法,但还需进一步优化。

AIM： To compare the cloning efficacy of full -length HBV genome amplified by single fragment PCR and two fragment PCR for choosing the suitable method for full - length HBV genome cloning. METHODS ： To amplify the fttll- length HBV genome from 85 sera sample of HBV patients, single fragment PCR and two fragment PCR were conducted. The products were cloned into the vector and sequenced after identified with double enzyme digestion. At the same time, the titers of 85 samples were detected by real - time PCR. RESULTS ： Compared with two fragment PCR, single fragment PCR requested higher level of sera HBV DNA for successful amplification of full - length HBV genome, and the efficacy of single fragment PCR was lower than that of two fragments PCR （ P 〈 0.05）. The mutation ratio of single fragment PCR was 1.13 bp/kb, and the sensitivity of single fragment PCR was 10^2 original templates. The efficacy of amplification was 80% if the amounts of template exceed 10^3, but the efficacy was low under this value. CONCLUSION： The efficacy of amplification is affected by the level of sera HBV DNA. The titers of sera HBV DNA are the proof for choosing a suitable PCR method. If the level of sera HBV DNA was more than 10^6 copies/L, single fragment PCR will be suitable. If the level of sera HBV DNA was less than 10^6 copies/L, two fragments PCR will be better.