Evaluation of dual priming oligonucleotide-based multiplex PCR for detection of HBV YMDD mutants

Archives of Virology - Tập 153 - Trang 2019-2025 - 2008
H. Y. Woo1, H. Park1, B. I. Kim2, W. K. Jeon2, Y. J. Kim3
1Department of Laboratory Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, South Korea
2Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, South Korea
3Department of Laboratory Medicine, Masan Samsung Hospital, Sungkyunkwan University School of Medicine, Masan, South Korea

Tóm tắt

We evaluated the usefulness of dual priming oligonucleotide (DPO)-based multiplex PCR, Seeplex HBV Lami-DR assay (Seegene Institute of Life Sciences, Seoul, Korea), to detect lamivudine-resistant HBV mutants in a comparison with the use of TRUGENE™ HBV genotyping and restriction fragment mass polymorphism (RFMP). Sera from 44 chronic hepatitis B patients were analyzed for the presence of mutations at codons 180 and 204 by performing DPO-based multiplex PCR, RFMP, and TRUGENE. The overall concordance rate among the three assays was 40.9% (18/44). Concordance rates between multiplex PCR and RFMP or multiplex PCR and TRUGENE were 61.4% (27/44) and 50.0% (22/44), respectively. In ten patients, multiplex PCR identified additional mutants not found using the other two methods. DPO-based multiplex PCR is a highly sensitive method to identify minor mutant populations and could be a practical tool in the monitoring of lamivudine resistance.

Tài liệu tham khảo

Allen M, Deslauriers M, Andrews C et al (1998) Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology 27:1670–1677 Liaw Y, Chien R, Yeh C et al (1999) Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy. Hepatology 30:567–572 Stuyver L, Locarnini S, Lok A et al (2001) Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region. Hepatology 33:751–757 Allen MI, Gauthier J, DesLauriers M et al (1999) Two sensitive PCR-based methods for detection of hepatitis B virus variants associated with reduced susceptibility to lamivudine. J Clin Microbiol 37:3338–3347 Jardi R, Buti M, Rodriguez-Frias F et al (1999) Rapid detection of lamivudine-resistant hepatitis B virus polymerase gene variants. J Virol Methods 83:181–187 Woo HY, Park H, Kim BI et al (2007) Comparison of mass spectrometric analysis and TRUGENE HBV genotyping for monitoring lamivudine resistance in chronic hepatitis B patients. Antivir Ther 12:7–13 Chun JY, Kim KJ, Hwang IT et al (2007) Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene. Nucleic Acids Res 35:e40 Kim JK, Lee HJ, Lee YJ et al (2008) Direct detection of lamivudine-resistant hepatitis B virus mutants by a multiplex PCR using dual-priming oligonucleotide primers. J Virol Methods 149:76–84 Roque-Afonso A, Ferey M, Mackiewicz V et al (2003) Monitoring the emergence of hepatitis B virus polymerase gene variants during lamivudine therapy in human immunodeficiency virus coinfected patients: performance of CLIPTM sequencing and line probe assay. Antivir Ther 8:627–634 Lampertico P, Vigano M, Manenti E et al (2005) Adefovir rapidly suppresses hepatitis B in HBeAg-negative patients developing genotypic resistance to lamivudine. Hepatology 42:1414–1419 Ohishi W, Shirakawa H, Kawakami Y et al (2004) Identification of rare polymerase variants of hepatitis B virus using a two-stage PCR with peptide nucleic acid clamping. J Med Virol 72:558–565 Krajden M, Comanor L, Rifkin O et al (1998) Assessment of hepatitis B virus DNA stability in serum by the Chiron Quantiplex branched-DNA assay. J Clin Microbiol 36:382–386 Jerome KR, Huang ML, Wald A et al (2002) Quantitative stability of DNA after extended storage of clinical specimens as determined by real-time PCR. J Clin Microbiol 40:2609–2611 Gutfreund KS, Williams M, George R et al (2000) Genotypic succession of mutations of the hepatitis B virus polymerase associated with lamivudine resistance. J Hepatol 33:469–475 Lee CH, Kim SO, Byun KS et al (2006) Predominance of hepatitis B virus YMDD mutants is prognostic of viral DNA breakthrough. Gastroenterology 130:1144–1152 Kim HS, Han KH, Ahn SH et al (2005) Evaluation of methods for monitoring drug resistance in chronic hepatitis B patients during lamivudine therapy based on mass spectrometry and reverse hybridization. Antivir Ther 10:441–449 Wen H, Li Q (2007) Real-time PCR detection of multiple lamivudine-resistant mutations with displacing probes in a single tube. J Clin Virol 38:334–340