Clinical Chemistry
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Clinical Chemistry 55: 1503-1509, 2009. First published June 18, 2009; 10.1373/clinchem.2009.124859
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(Clinical Chemistry. 2009;55:1503-1509.)
© 2009 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Multiplex Detection of 60 Hepatitis B Virus Variants by MALDI-TOF Mass Spectrometry

Ju Luan1,2, Jing Yuan2,2, Xiaohe Li2, Shengnan Jin1, Ling Yu1, Mingfeng Liao2, Hongmei Zhang2, Cheng Xu2, Qing He2, Bin Wen2, Xunhua Zhong2, Xinchun Chen2, Henry L.Y. Chan3, Joseph J.Y. Sung3, Boping Zhou2,a and Chunming Ding4,a

1 Stanley Ho Centre for Emerging Infectious Diseases and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR; 2 Shenzhen-Hongkong Institute of infectious diseases, The Third People’s Hospital Of Shenzhen, Shenzhen, Guangdong, P.R. China; 3 Department of Medicine and Therapeutics, Institute of Digestive Disease, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR; 4 Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore.

aAddress correspondence to: C.D. at Brenner Centre for Molecular Medicine, 30 Medical Dr., Singapore 117609. Fax +65-6776-6840; e-mail cmding{at}gmail.com. B.Z. at Shenzhen Donghu Hospital, Shenzhen, Guangdong 518020, P.R. China. E-mail zhoubp{at}hotmail.com.

Background: Variations in the hepatitis B virus (HBV) genome may develop spontaneously or under selective pressure from antiviral therapy. Such variations may confer drug resistance or affect virus replication capacity, resulting in failure of antiviral therapy.

Methods: A duplex PCR was used to amplify the region of the reverse transcriptase gene, the precore promoter, and the basal core promoter of the HBV genome. Four multiplex primer-extension reactions were used to interrogate 60 frequently observed HBV variants during antiviral therapy. Automated MALDI-TOF mass spectrometry (MS) was used for mutation detection. Capillary sequencing was used to confirm the MS results.

Results: The limit of quantification was 1000 HBV copies/mL for multiplex detection of HBV variants. Fifty-three variants (88.3%) were analyzed successfully in at least 90% of the sera from 88 treatment-naive patients and 80 patients with virologic breakthrough. MS was able to detect twice as many minor variants as direct sequencing while achieving close to full automation. MS and direct sequencing showed only 0.1% discordance in variant calls.

Conclusions: This platform based on multiplex primer extension and MALDI-TOF MS was able to detect 60 HBV variants in 4 multiplex reactions with accuracy and low detection limits.






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Copyright © 2009 by the American Association for Clinical Chemistry.