Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays

Shuang Meng,Sien Zhan,Jinming Li

doi:10.1186/1743-422x-6-226

Abstract

BackgroundIdentical blood samples tested using different kits can give markedly different hepatitis B virus (HBV) DNA levels, which can cause difficulty in the interpretation of viral load. A universal double-stranded DNA control or standard that can be used in all commercial HBV DNA nucleic acid amplification assay kits is urgently needed. By aligning all HBV genotypes (A-H), we found that the surface antigen gene and precore-core gene regions of HBV are the most conserved regions among the different HBV genotypes. We constructed a chimeric fragment by overlapping extension polymerase chain reaction and obtained a 1,349-bp HBVC+S fragment. We then packaged the fragment into lambda phages using a traditional lambda phage cloning procedure.ResultsThe obtained armored DNA was resistant to DNase I digestion and was stable, noninfectious to humans, and could be easily extracted using commercial kits. More importantly, the armored DNA may be used with all HBV DNA nucleic acid amplification assay kits.ConclusionsThe lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard. In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits. Thus, the constructed armored DNA can probably be used as a universal positive control or standard in HBV DNA assays.

Highlights

Identical blood samples tested using different kits can give markedly different hepatitis B virus (HBV) DNA levels, which can cause difficulty in the interpretation of viral load
Identification of Armored DNA by Polymerase Chain Reaction and Sequencing The DNA extracted from lambda phages was amplified with the phage-specific primers gt11-for and gt11-rev
The result indicated that the sequencing of the polymerase chain reaction (PCR) products was accurate and the recombinant plasmid gt11-HBVC+S were successfully packaged by the phage packaging extracts

Summary

Introduction

Identical blood samples tested using different kits can give markedly different hepatitis B virus (HBV) DNA levels, which can cause difficulty in the interpretation of viral load. Hepatitis B virus (HBV) infection is a major public health problem It is responsible for chronic liver disease and is a risk factor for liver cirrhosis and hepatocellular carcinoma [1,2,3]. Real-time polymerase chain reaction (PCR) assays are widely used for the detection and quantification of HBV DNA in clinical samples [4,5,6]. All of the HBV DNA nucleic acid amplification detection kits target the highly conserved surface antigen gene or the precore-core genes of the HBV genome [10,12,13,14,15,16,17] and the HBV DNA controls or standards come from many sources [18,19,20]

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Discussion

Conclusion