Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B

AIM： To compare the sequencing of PCR products, pyrosequencing, and real-time PCR for detection of Tyrosinemethionine-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...

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Published in:World journal of gastroenterology : WJG 2006-11, Vol.12 (44), p.7192-7196
Main Authors: Yang, Zhi-Jun, Tu, Mei-Zeng, Liu, Jian, Wang, Xiao-Ling, Jin, Hong-Zhi
Format: Article
Language:English
Subjects:
Drug Resistance, Viral - genetics
Genetic Techniques - economics
Hepacivirus - chemistry
Hepacivirus - genetics
Hepatitis B, Chronic - drug therapy
Hepatitis B, Chronic - virology
Humans
Lamivudine - therapeutic use
PCR
Plasmids - chemistry
Polymerase Chain Reaction
Rapid Communication
Reverse Transcriptase Inhibitors - therapeutic use
RNA-Directed DNA Polymerase - chemistry
RNA-Directed DNA Polymerase - genetics
Sequence Analysis, DNA
Time Factors
乙型肝炎
序列测定
突变体
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Summary:AIM： To compare the sequencing of PCR products, pyrosequencing, and real-time PCR for detection of Tyrosinemethionine-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.
ISSN:1007-9327
2219-2840
DOI:10.3748/wjg.v12.i44.7192