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World J Gastroenterol. Nov 28, 2006; 12(44): 7192-7196
Published online Nov 28, 2006. doi: 10.3748/wjg.v12.i44.7192
Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B
Zhi-Jun Yang, Mei-Zeng Tu, Jian Liu, Xiao-Ling Wang, Hong-Zhi Jin
Zhi-Jun Yang, Mei-Zeng Tu, Management School, Shanghai Jiaotong University, Shanghai 200052, China
Jian Liu, Laboratory Center, First Hospital of Yangzhou, Yangzhou 225001, Jiangsu Province, China
Xiao-Ling Wang, Laboratory Center, Subei People’s Hospital, Yangzhou 225001, Jiangsu Province, China
Hong-Zhi Jin, School of Life Science, Shanghai Jiaotong University, Shanghai 200240, China
Correspondence to: Zhi-Jun Yang, PhD, Management School, Shanghai Jiaotong University, No. 1289 Yishan Road, Shanghai 200233, China. yangzj@fosun.com.cn
Telephone: +86-21-64952059 Fax: +86-21-64955476
Received: September 18, 2006
Revised: September 28, 2006
Accepted: October 20, 2006
Published online: November 28, 2006
Abstract

AIM: To compare the sequencing of PCR products, pyrosequencing, and real-time PCR for detection of Tyrosine-methionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B.

METHODS: Mixtures of plasmids and serum samples from 69 chronic hepatitis B patients treated with lamivudine were tested for YMDD mutations by sequencing of PCR products, pyrosequencing, and real-time PCR, respectively. Time required and reagent costs of the three assays were evaluated.

RESULTS: Real-time PCR detected 100%, 50%, 10%, 1% and 0.1% of YVDD plasmid in mixtures with 106 copies/mL of YMDD plasmid, whereas sequencing and pyrosequencing only detected 100% and 50% of YVDD plasmid in aliquots of the corresponding mixtures. Completely concordant results were obtained from 60 (87%) out of the 69 clinical serum samples by the three assays. Mutants were detected by real-time PCR in less than 20% of the total virus population, but no mutant was detected by sequencing and pyrosequencing. In addition, real-time PCR required less time and was more cost-effective than the other two assays. However, throughput of pyrosequencing was the highest.

CONCLUSION: Among the three assays compared, real-time PCR is the most sensitive, cost-effective, and time saving for monitoring YMDD mutants in patients with chronic hepatitis B on lamivudine therapy.

Keywords: Tyrosine-methionine-aspartate-aspartate mutant; Hepatitis B virus; Real-time PCR; Sequencing; Pyrosequencing