Ultrasensitive quantification of hepatitis B virus A1762T/G1764A mutant by a SimpleProbe PCR using a wild type-selective PCR blocker and a primer-blocker-probe partial overlap approach

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http://jcm.asm.org/cgi/content/abstract/JCM.02472-10v1

JCM Accepts, published online ahead of print on 11 May 2011

J. Clin. Microbiol. doi:10.1128/JCM.02472-10

Copyright © 2011,American Society for Microbiology and/or the Listed

Authors/Institutions. .

Ultrasensitive quantification of hepatitis B virus A1762T/G1764A mutant by a

SimpleProbe PCR using a wild type-selective PCR blocker and a

primer-blocker-probe partial overlap approach

Hui Nie1, Alison A. 2,3, W. London4, M. Block1,3,5 and

Xiangdong Ren1,5,6,*

1 Department of Microbiology and Immunology, Drexel University College of

Medicine, Doylestown, PA

2 School of Public Health, Drexel University, Philadelphia, PA

3 Hepatitis B Foundation, Doylestown, PA

4 Fox Chase Cancer Center, Philadelphia, PA

5 Institute for Hepatitis and Virus Research, Doylestown, PA

6 Reniguard Life Sciences Inc, Doylestown, PA

* Corresponding Author: Mailing Address: Institute for Hepatitis and Virus

Research, 3805 Old Easton Road, Doylestown, PA 18902, USA. Telephone: (215)

589-6357; Fax: (215) 489-4920; Email: drren001@...

Hepatitis B virus (HBV) carrying the A1762T/G1764A double mutation in the basal

core promoter (BCP) region is associated with HBe antigen seroconversion and

increased risk of liver cirrhosis and hepatocellular carcinoma (HCC).

Quantification of the mutant viruses may help predicting the risk of HCC.

However, the viral genome tends to have nucleotide polymorphism which makes it

difficult to design hybridization-based assays including real time PCR.

Ultrasensitive quantification of the mutant viruses at the early developmental

stage is even more challenging as the mutant is masked by excessive amount of

the wild type (WT) viruses. In this study, we developed a selective inhibitory

PCR (siPCR) using a locked nucleic acid-based PCR blocker to selectively inhibit

the amplification of the WT viral DNA but not the mutant DNA. At the end of

siPCR, the proportion of the mutant could be increased by about 10,000 fold,

making the mutant more readily detectable by downstream applications such as

real time PCR and DNA sequencing. We also describe a primer-probe partial

overlap approach which significantly simplified the melting curve patterns and

minimized the influence of viral genome polymorphism on assay accuracy. Analysis

of 62 patient samples showed complete match of the melting curve patterns with

the sequencing results. More than 97% of HBV BCP sequences in the GenBank can be

correctly identified by the melting curve analysis. Combination of siPCR and the

SimpleProbe real time PCR enabled mutant quantification in the presence of

100,000-fold excess of the WT DNA.