A method for rapid detection and genotype identification of hepatitis C virus 1–6 by one-step reverse transcription loop-mediated isothermal amplification

Abstract

Objective: Hepatitis C virus (HCV) is probably the leading cause of liver cirrhosis and hepatocellular carcinoma globally. Diagnostic tools conventionally used for the detection and identification of HCV infection are technically demanding, time-consuming, and costly for resource-limited environments. This study reports the development of the first rapid loop-mediated reverse transcription isothermal amplification assay that rapidly detects and identifies HCV genotypes in blood components. Methods: RNA extracted from donor plasma and serum specimens was applied to a one-step reverse transcription loop-mediated isothermal amplification reaction performed with HCV-specific oligonucleotides. Reactions were conducted at 63.5 °C for 30–60 min. The diagnostic characteristics of the assay were investigated and validated with clinical specimens. Results: Electrophoretic analysis of amplification revealed detection and identification of HCV genotypes 1–6. Positive amplification revealed unique ladder-like banding patterns that identified each HCV genotype. The assay demonstrated a sensitivity of 91.5% and specificity of 100%. Rapid naked-eye detection of HCV infection was facilitated by observation of an intense fluorescent glow of amplified targets under UV illumination. Conclusion: These diagnostic characteristics highlight the potential utility of this assay for the rapid detection and genotype identification of HCV infection in field and point-of-care settings in endemic regions and resource-limited environments.

Keywords

• Hepatitis C virus
• Detection
• Isothermal amplification
• Genotype identification
• Plasma
• Serum