Dougbeh-Chris Nyan1, Kevin L Swinson2. 1. Division of Emerging and Transfusion-Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Ave, Silver Spring, MD 20993, USA. Electronic address: dnyan@doctor.com. 2. Department of Biology, Morgan State University, Baltimore, Maryland, USA.
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.
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.
Authors: Weronika Witkowska McConnell; Chris Davis; Suleman R Sabir; Alice Garrett; Amanda Bradley-Stewart; Pawel Jajesniak; Julien Reboud; Gaolian Xu; Zhugen Yang; Rory Gunson; Emma C Thomson; Jonathan M Cooper Journal: Nat Commun Date: 2021-11-30 Impact factor: 14.919