The Effectiveness Evaluation On Plasmid In Internal Quality Control Of HBV DNA

Objective Based on its high specificity and sensitivity, fluores-cence quantitative PCR has been widely used in the HBV DNA quantitative detection and plays an important role in the diagnosis, treatment and prognosis of Hepatitis B. However, it is internal quality control that ensure accurate and reliable test results of fluores-cence quantitative PCR. In this study, self-made internal quality control material has been obtained by using genetic engineering methods and its effectiveness in clinical routine work has also been evaluated.Methods A total of 65 HBV genome sequence are selected from GeneBank database, covering eight genotypes of HBV from A to H. After multiple sequence alignment of each genotype of HBV by Clustal X2 software, PCR primers are designed according to conserved HBV S region. The target HBV DNA fragments have been subsequently obtained by PCR amplification and then inserted pEASY-T1 to construct the recombinant plasmid vector. After that, two levels of internal quality control materials(107copies / ml,104 copies / ml) have been made from serum-diluted recombinant plasmid. Finally, the target value and precision of self-made internal quality control materials are confirmed.Results The target HBV S gene fragments have been correctly inserted into pEASY-T1. NCBI BLAST sequence analysis show that the consistent rate of obtained HBV S gene fragments and complementary fragments in GenBank is 99%, without similarity to the known human and the other viral DNA sequence. The evaluation of two levels of self-control materials has also found that Intra-assay precision(CV) are 2.16%, 2.76% and inter-assay precision(CV) are 2.27%, 3.19%. Repeatability and stability of the self-made internal quality control material are also in line with the requirements of the experiment.Conclusions By genetic engineering techniques, recombinant HBV S region plasmid has been successfully obtained and new internal quality control material then be synthesized. Self-made internal quality control material is simple and stable which can be used as internal quality control of HBV DNA quantitative detection.