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


RESULTS
The 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.


CONCLUSIONS
The 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.
Meng
Shuang
S
National Center for Clinical Laboratories, Beijing Hospital, Beijing, PR China.xluhua@163.com
Zhan
Sien
S
Li
Jinming
J
D010582:Bacteriophage lambda / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000097:blood / Q000235:genetics / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D006512:Hepatitis B Core Antigens / Q000235:genetics; D006514:Hepatitis B Surface Antigens / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics / Q000502:physiology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000592:standards*; D011786:Quality Control; D011933:Reagent Kits, Diagnostic; D012015:Reference Standards; D019562:Viral Load
D004279:DNA, Viral; D006512:Hepatitis B Core Antigens; D006514:Hepatitis B Surface Antigens; D011933:Reagent Kits, Diagnostic; D004247:DNA; D003851:Deoxyribonucleases