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

Meng S, Zhan S, Li J - Virol. J. (2009)

Bottom Line: 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.The lambda phage packaging system can be used as an excellent expression platform for armored DNA.In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Center for Clinical Laboratories, Beijing Hospital, Beijing, PR China.xluhua@163.com

ABSTRACT

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.

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Related in: MedlinePlus

Identification of Armored DNA by PCR Amplification. The PCR amplification products of the DNA extracted from lambda phages were analysed using ethidium-bromide stained 1% agarose gel. Lane 1: negative control with no template; Lane 2, 3, 4, 5, and 6: PCR products from positive chimeric phages.
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Figure 2: Identification of Armored DNA by PCR Amplification. The PCR amplification products of the DNA extracted from lambda phages were analysed using ethidium-bromide stained 1% agarose gel. Lane 1: negative control with no template; Lane 2, 3, 4, 5, and 6: PCR products from positive chimeric phages.

Mentions: The DNA extracted from lambda phages was amplified with the phage-specific primers gt11-for and gt11-rev. The resulting PCR products were analysed by electrophoresis on agarose gel (1%) containing ethidium bromide (Fig. 2) and sequencing. The result indicated that the sequencing of the PCR products was accurate and the recombinant plasmid gt11-HBVC+S were successfully packaged by the phage packaging extracts.


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

Meng S, Zhan S, Li J - Virol. J. (2009)

Identification of Armored DNA by PCR Amplification. The PCR amplification products of the DNA extracted from lambda phages were analysed using ethidium-bromide stained 1% agarose gel. Lane 1: negative control with no template; Lane 2, 3, 4, 5, and 6: PCR products from positive chimeric phages.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803455&req=5

Figure 2: Identification of Armored DNA by PCR Amplification. The PCR amplification products of the DNA extracted from lambda phages were analysed using ethidium-bromide stained 1% agarose gel. Lane 1: negative control with no template; Lane 2, 3, 4, 5, and 6: PCR products from positive chimeric phages.
Mentions: The DNA extracted from lambda phages was amplified with the phage-specific primers gt11-for and gt11-rev. The resulting PCR products were analysed by electrophoresis on agarose gel (1%) containing ethidium bromide (Fig. 2) and sequencing. The result indicated that the sequencing of the PCR products was accurate and the recombinant plasmid gt11-HBVC+S were successfully packaged by the phage packaging extracts.

Bottom Line: 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.The lambda phage packaging system can be used as an excellent expression platform for armored DNA.In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Center for Clinical Laboratories, Beijing Hospital, Beijing, PR China.xluhua@163.com

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus