Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B.
OBJECTIVE
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 10(6) 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.


CONCLUSIONS
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.
Yang
Zhi-Jun
ZJ
Management School, Shanghai Jiaotong University, No. 1289 Yishan Road, Shanghai 200233, China. yangzj@fosun.com.cn
Tu
Mei-Zeng
MZ
Liu
Jian
J
Wang
Xiao-Ling
XL
Jin
Hong-Zhi
HZ
D024882:Drug Resistance, Viral / Q000235:genetics*; D005821:Genetic Techniques / Q000191:economics*; D016174:Hepacivirus / Q000737:chemistry* / Q000235:genetics; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D019259:Lamivudine / Q000627:therapeutic use; D010957:Plasmids / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use; D017422:Sequence Analysis, DNA; D013997:Time Factors
D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine; D012194:RNA-Directed DNA Polymerase