Rapid and accurate genotyping of YMDD motif variants in the hepatitis B virus genome by an improved reverse dot blot method.
By using the "flow-through hybridization" principle, we developed a new, rapid and accurate reverse dot blot (RDB) method to detect lamivudine resistance-associated YMDD motif variants in hepatitis B virus (HBV) genome. The improved RDB method was very fast at simultaneously detecting HBV YMDD wild-type and mutant motifs. In a blind analysis, 100 samples previously genotyped by DNA clonal sequencing analysis were used to evaluate the sensitivity and specificity of this assay. Conventional restriction fragment length polymorphism (RFLP) data were also used to test our method. In blind experiments, our improved RDB method had an accuracy and specificity of 100%, which was much higher than RFLP, which had an accuracy and specificity of only 83.0%. In clinical detection practice, 49 patients highly suspected of lamivudine resistance were successfully diagnosed by this method. Our improved RDB assay is a simple, rapid, cheap, semiautomatic, accurate, sensitive, and contamination-proof method of detecting lamivudine resistance-associated mutants in the human hepatitis B virus genome.
Ou
Zhi-Ying
ZY
Department of Anatomy, Zhongshan Medical School of Sun Yat-Sen University, 74 Zhongshan Rd. 2, Guangzhou, People's Republic of China 510080. ouzhiying@yahoo.com.cn
Liu
Na
N
Chen
Chang-Jie
CJ
Cheng
Gang
G
He
Yun-Shao
YS
D020816:Amino Acid Motifs / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D024882:Drug Resistance, Viral; D016679:Genome, Viral; D006509:Hepatitis B / Q000175:diagnosis; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology*; D009154:Mutation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D012680:Sensitivity and Specificity
D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine; D004259:DNA-Directed DNA Polymerase