Development of a novel genotype-specific loop-mediated isothermal amplification technique for Hepatitis B virus genotypes B and C genotyping and quantification

Background: There is the need for a rapid, economical method for genotyping Hepatitis B virus (HBV) to support clinical practice. Objectives: To develop a novel HBV genotyping process using genotype specific loop mediated isothermal amplification (LAMP). Study design: HBV genotypes B and C specific LAMP methods were evaluated using standard panel. A comparative analysis of the LAMP test against Taqman assay using 105 clinical samples, was undertaken to evaluate the quantitation capacity of the method. 111 clinical samples were used to test the clinical applicability of the genotype specific LAMP method. The results were compared with those obtained by real-time PCR based genotyping and sequencing. Results: Using genotype-specific primers, the LAMP assay correctly identified all predefined genotypes B and C, and no cross-reaction was observed. Real-time format of this assay provides simultaneous identification and quantification of genotypes B and C. The detection sensitivity of the method was found to be 323 and 515 copies/ml for genotypes B and C specific LAMP assay respectively. High correlation (R-2 = 0.91) and good agreement between the LAMP method and the real-time PCR test were achieved for HBV quantitation. Samples from 111 HBV-infected patients were genotyped with LAMP, revealing 53% HBV as genotype B, 36% as genotype C, and 12% as mixed genotypes B and C. LAMP method showed coincidence rates of 96.7% with the real-time PCR genotyping results. Conclusion: This approach is a promising tool for HBV genotyping and quantitation. It appears to be useful for routine clinical practice even in field investigation. (C) 2011 Elsevier B.V. All rights reserved.