Distinguishing Four HCV Genotypes by Loop Mediated Isothermal Amplification Assay.

Background: Genotyping of hepatitis C virus (HCV) is of great significance for clinical diagnosis and treatment. The object of this work was to establish a loop-mediated isothermal amplification (LAMP) method for four subtypes of HCV prevalent in China.

Methods: Gene sequences of HCV-1b, 2a, 3, and 6a were downloaded from Genebank. Primer sets of the four HCV subtypes for LAMP method were designed by Primer Explorer V.4. LAMP reactions were performed at 65°C for 60 minutes. Sera of HCV-1b, 2a, 3, and 6a, HBV-A, HBV-B, HIV-1, HIV-2 and HAV, synthesized nucleic acid of HCV-4a, and 5a were used for evaluation of LAMP performance. For specificity evaluation, each primer set for HCV-1b, 2a, 3, and 6a was tested with all kinds of sera. Each serum of the above 4 HCV subtypes was diluted and aliquoted into 10 parts. RNA was subsequently extracted, which was detected by LAMP and Real-time PCR to verify limit of detection (LOD).

Results: The LAMP assay established in this study could detect and distinguish HCV-1b, 2a, 3, and 6a. Consistent with gene sequencing methods, specificity of HCV-1b, 2a, 3, and 6a was 100%, 85%, 90%, and 100% detected by LAMP, respectively. The LOD of HCV-1b, 3, and 6a was 1.5 x 103 IU/mL. The LOD of HCV2a was 1.0 x 103 IU/mL with the LAMP method.

Conclusions: The LAMP method established in this study is feasible for distinguishing HCV-1b, 2a, 3, and 6a specifically and sensitively.