Quantification of complex precore mutations of hepatitis B virus by SimpleProbe real time PCR and dual melting analysis

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http://www.journalofclinicalvirology.com/article/PIIS1386653211001922/abstract?r\

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JOURNAL OF CLINICAL VIROLOGY

Articles in Press

Quantification of complex precore mutations of hepatitis B virus by SimpleProbe

real time PCR and dual melting analysis

Articles in Press

Quantification of complex precore mutations of hepatitis B virus by SimpleProbe

real time PCR and dual melting analysis

Hui Niea Alison A. bc, W. Londond, M. Blockace, Xiangdong

Renaef

Received 6 February 2011; received in revised form 20 May 2011; accepted 20 May

2011. published online 13 June 2011.

Corrected Proof

Abstract

Background

Hepatitis B virus (HBV) precore G1896A mutation is associated with Hepatitis B e

antigen (HBeAg) seroconversion. This mutation and the adjacent G1899A mutation

also appear to associate with increased risk of hepatocellular carcinoma.

Quantitative mutant dynamics may help determine the potential of these mutants

as clinical biomarkers. However, a reliable method to quantify either mutant is

not available, partly because the viral genome has polymorphisms in general and

the precore mutations are complex.

Objectives

(1) To develop a reliable and ultrasensitive assay for the quantification of HBV

G1896A and/or G1899A mutants. (2) To obtain preliminary data on the quantities

of the precore mutants in patients.

Study design

A SimpleProbe real time PCR assay was developed to quantify the HBV precore

mutants. Dual melting analysis and a primer-probe partial overlap approach were

used to increase detection accuracy. A wild-type selective PCR blocker was also

developed to increase mutant detection sensitivity.

Results

The assay correctly identified the precore sequence from all 62 patient samples

analyzed. More than 97% of precore sequences in the GenBank can be recognized.

Mutant detection sensitivity reached 0.001% using a wild type-selective PCR

blocker. At least one precore mutant can be detected from all 20 HBeAg-positive

individuals who were negative for precore mutations by DNA sequencing.

Conclusions

The reliability of this ultrasensitive mutation quantification assay was

demonstrated. The same approaches may be useful for the detection of other

clinically significant mutations. Evolution of the precore mutants warrants

further studies.

Abbreviations: HBV, hepatitis B virus, HBeAg, hepatitis B e antigen, HCC,

hepatocellular carcinoma, LNA, locked nucleic acid, nt, nucleotide, TVL, total

viral load, WT, wild type

a Department of Mcrobiology and Immunology, Drexel University College of

Medicine, Doylestown, PA 18902, United States

b School of Public Health, Drexel University, Philadelphia, PA 19102, United

States

c Hepatitis B Foundation, Doylestown, PA 18902, United States

d Fox Chase Cancer Center, Philadelphia, PA 19111, United States

e Institute for Hepatitis and Virus Research, Doylestown, PA 18902, United

States

f Reniguard Life Sciences Inc, Doylestown, PA 18902, United States

Corresponding author at: Institute for Hepatitis and Virus Research, 3805 Old

Easton Road, Doylestown, PA 18902, United States. Tel.: +1 215 589 6357; fax: +1

215 489 4920.

PII: S1386-6532(11)00192-2

doi:10.1016/j.jcv.2011.05.020

© 2011 Elsevier B.V. All rights reserved.

[Guest guest]

http://www.journalofclinicalvirology.com/article/PIIS1386653211001922/abstract?r\

ss=yes

JOURNAL OF CLINICAL VIROLOGY

Articles in Press

Quantification of complex precore mutations of hepatitis B virus by SimpleProbe

real time PCR and dual melting analysis

Articles in Press

Quantification of complex precore mutations of hepatitis B virus by SimpleProbe

real time PCR and dual melting analysis

Hui Niea Alison A. bc, W. Londond, M. Blockace, Xiangdong

Renaef

Received 6 February 2011; received in revised form 20 May 2011; accepted 20 May

2011. published online 13 June 2011.

Corrected Proof

Abstract

Background

Hepatitis B virus (HBV) precore G1896A mutation is associated with Hepatitis B e

antigen (HBeAg) seroconversion. This mutation and the adjacent G1899A mutation

also appear to associate with increased risk of hepatocellular carcinoma.

Quantitative mutant dynamics may help determine the potential of these mutants

as clinical biomarkers. However, a reliable method to quantify either mutant is

not available, partly because the viral genome has polymorphisms in general and

the precore mutations are complex.

Objectives

(1) To develop a reliable and ultrasensitive assay for the quantification of HBV

G1896A and/or G1899A mutants. (2) To obtain preliminary data on the quantities

of the precore mutants in patients.

Study design

A SimpleProbe real time PCR assay was developed to quantify the HBV precore

mutants. Dual melting analysis and a primer-probe partial overlap approach were

used to increase detection accuracy. A wild-type selective PCR blocker was also

developed to increase mutant detection sensitivity.

Results

The assay correctly identified the precore sequence from all 62 patient samples

analyzed. More than 97% of precore sequences in the GenBank can be recognized.

Mutant detection sensitivity reached 0.001% using a wild type-selective PCR

blocker. At least one precore mutant can be detected from all 20 HBeAg-positive

individuals who were negative for precore mutations by DNA sequencing.

Conclusions

The reliability of this ultrasensitive mutation quantification assay was

demonstrated. The same approaches may be useful for the detection of other

clinically significant mutations. Evolution of the precore mutants warrants

further studies.

Abbreviations: HBV, hepatitis B virus, HBeAg, hepatitis B e antigen, HCC,

hepatocellular carcinoma, LNA, locked nucleic acid, nt, nucleotide, TVL, total

viral load, WT, wild type

a Department of Mcrobiology and Immunology, Drexel University College of

Medicine, Doylestown, PA 18902, United States

b School of Public Health, Drexel University, Philadelphia, PA 19102, United

States

c Hepatitis B Foundation, Doylestown, PA 18902, United States

d Fox Chase Cancer Center, Philadelphia, PA 19111, United States

e Institute for Hepatitis and Virus Research, Doylestown, PA 18902, United

States

f Reniguard Life Sciences Inc, Doylestown, PA 18902, United States

Corresponding author at: Institute for Hepatitis and Virus Research, 3805 Old

Easton Road, Doylestown, PA 18902, United States. Tel.: +1 215 589 6357; fax: +1

215 489 4920.

PII: S1386-6532(11)00192-2

doi:10.1016/j.jcv.2011.05.020

© 2011 Elsevier B.V. All rights reserved.