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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

Verification of the Recombinant pGEM-HBVC+S Plasmid. The PCR amplification products from recombinant pGEM-HBVc+s were analysed using ethidium-bromide-stained 1% agarose gel. Lanes 1, 2, and 3: negative control with no template; Lane 4: positive control of recombinant clones; Lane 5 and 6: PCR products of positive recombinant clones.
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Figure 1: Verification of the Recombinant pGEM-HBVC+S Plasmid. The PCR amplification products from recombinant pGEM-HBVc+s were analysed using ethidium-bromide-stained 1% agarose gel. Lanes 1, 2, and 3: negative control with no template; Lane 4: positive control of recombinant clones; Lane 5 and 6: PCR products of positive recombinant clones.

Mentions: The PCR products from the recombinant pGEM- HBVC+S (using primers 1 and 4) were full length (1,349 bp; Fig. 1). Sequencing also demonstrated that the inserted HBV sequence was 1,349 bp without point mutation. Thus, the recombinant plasmid pGEM- HBVC+S was successfully constructed.


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)

Verification of the Recombinant pGEM-HBVC+S Plasmid. The PCR amplification products from recombinant pGEM-HBVc+s were analysed using ethidium-bromide-stained 1% agarose gel. Lanes 1, 2, and 3: negative control with no template; Lane 4: positive control of recombinant clones; Lane 5 and 6: PCR products of positive recombinant clones.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Verification of the Recombinant pGEM-HBVC+S Plasmid. The PCR amplification products from recombinant pGEM-HBVc+s were analysed using ethidium-bromide-stained 1% agarose gel. Lanes 1, 2, and 3: negative control with no template; Lane 4: positive control of recombinant clones; Lane 5 and 6: PCR products of positive recombinant clones.
Mentions: The PCR products from the recombinant pGEM- HBVC+S (using primers 1 and 4) were full length (1,349 bp; Fig. 1). Sequencing also demonstrated that the inserted HBV sequence was 1,349 bp without point mutation. Thus, the recombinant plasmid pGEM- HBVC+S was successfully constructed.

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