乙型肝炎病毒DNA定量检测室内质控品的制备及效果评价

Preparation and effectiveness evaluation on internal quality control for quantitative determination of hepatitis B virus DNA

目的制备乙型肝炎病毒（hepatitis B virus,HBV）DNA定量检测室内质控品,并进行初步评价。方法对HBV各个基因型序列进行多序列比对分析,选择保守序列S区设计引物,PCR扩增HBV S区基因,与pEASY-T1载体连接,构建重组质粒。测序后,将重组质粒稀释成10~7 copies/ml、10~4 copies/ml两个水平,分别作为高值质控品（HBVH）和低值质控品（HBV-L）,分装冷冻于-80℃,并初定靶值。评价自制质控品的精密度和稳定性,绘制室内质控图。结果构建的重组质粒测序结果与目的片段一致,自制HBV DNA 2个水平室内质控品的精密度、稳定性均达到实验要求。结论自制HBV DNA 2个水平的质控品可用于HBV DNA定量检测的室内质控。

Objective To prepare and preliminarily evaluate the internal quality control for quantitative determination of hepatitis B virus （HBV） DNA. Methods Multiple sequence alignment analysis was performed on HBV genotypes, and primers were designed based on the conserved region of HBV S gene for amplification by PCR. The PCR products were inserted into vector pEASY-T1, and the constructed recombinant plasmids were identified by sequencing, then diluted to concentrations of 107 and 104 copies/ml and used as high （HBV-H） and low （HBV-L） HBV DNA level quality controls respectively. The quality controls were stored at -80 ℃in frozen and, after the initial target value was confirmed, evaluated for precision and stability, of which the internal quality control chart was plotted. Results The result of DNA sequencing of constructed recombinant plasmid was consistent with that of target fragment. Both the precision and stability of prepared internal quality control met the experimental requirements. Conclusion The prepared internal quality controls may be used for internal quality control of quantitative determination of HBV DNA.