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Journal of Clinical Microbiology, November 2005, p. 5685-5689, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5685-5689.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid and Accurate Genotyping of YMDD Motif Variants in the Hepatitis B Virus Genome by an Improved Reverse Dot Blot Method

Zhi-Ying Ou,1,2 Na Liu,1,2 Chang-Jie Chen,1,2 Gang Cheng,2 and Yun-Shao He1,2*

Department of Anatomy,1 DaAn Gene Diagnostic Center, Zhongshan Medical College of Sun Yat-Sen University, Guangzhou, Guangdong, People's Republic of China2

Received 4 March 2005/ Returned for modification 2 May 2005/ Accepted 12 August 2005

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.

* Corresponding author. Mailing address: DaAn Gene Diagnostic Center, Zhongshan Medical School of Sun Yat-Sen University, 74 Zhongshan Rd. 2, Guangzhou, People's Republic of China 510080. Phone: 86 20 88288735. Fax: 86 20 8733 3435. E-mail: ouzhiying{at}yahoo.com.cn.

Journal of Clinical Microbiology, November 2005, p. 5685-5689, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5685-5689.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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