Sayamon Hongjaisee1, Natteewan Doungjinda2, Woottichai Khamduang3, Tanawan Samleerat Carraway3, Jiraprapa Wipasa2, Jose D Debes4, Khuanchai Supparatpinyo2. 1. Research Institute for Health Sciences, Chiang Mai University, Chiangmai, Thailand. Electronic address: sayamon.ho@cmu.ac.th. 2. Research Institute for Health Sciences, Chiang Mai University, Chiangmai, Thailand. 3. Infectious Disease Research Unit, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiangmai, Thailand. 4. Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA.
Abstract
OBJECTIVES: The aim was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of hepatitis C virus (HCV) in a single closed tube. METHODS: Plasma samples were collected from 200 HCV-infected patients. HCV-RNA was detected by one-step RT-LAMP processed at 65 °C for 60 min. The amplified products were detected by hydroxynaphthol blue (HNB)-dependent visual method and gel electrophoresis. Specificity was tested against other viruses. Sensitivity was determined using serial dilutions of extracted RNA. RESULTS: The RT-LAMP assay detected 97.5% of HCV-RNA genotype 1, 91.1% of genotype 3, and 100% of genotype 6. The color change was evidenced with the naked eye. The assay demonstrated a clinical sensitivity of 95.5% and specificity of 100%, as well as no cross-reactivity with other viruses (i.e., hepatitis B virus, HIV). The limit of detection was as low as 10 ng per reaction for HCV genotypes 1a and 6, while it was 100 ng for genotype 3a. The assay showed a 100% detection threshold at a viral load of 5.00 log10 IU/mL in the clinical samples tested. CONCLUSIONS: This study demonstrated the use of an RT-LAMP assay for the detection of HCV in a simple, rapid, and cost-effective manner, which will be useful in resource-limited settings to allow the identification of individuals in need of HCV treatment.
OBJECTIVES: The aim was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of hepatitis C virus (HCV) in a single closed tube. METHODS: Plasma samples were collected from 200 HCV-infectedpatients. HCV-RNA was detected by one-step RT-LAMP processed at 65 °C for 60 min. The amplified products were detected by hydroxynaphthol blue (HNB)-dependent visual method and gel electrophoresis. Specificity was tested against other viruses. Sensitivity was determined using serial dilutions of extracted RNA. RESULTS: The RT-LAMP assay detected 97.5% of HCV-RNA genotype 1, 91.1% of genotype 3, and 100% of genotype 6. The color change was evidenced with the naked eye. The assay demonstrated a clinical sensitivity of 95.5% and specificity of 100%, as well as no cross-reactivity with other viruses (i.e., hepatitis B virus, HIV). The limit of detection was as low as 10 ng per reaction for HCV genotypes 1a and 6, while it was 100 ng for genotype 3a. The assay showed a 100% detection threshold at a viral load of 5.00 log10 IU/mL in the clinical samples tested. CONCLUSIONS: This study demonstrated the use of an RT-LAMP assay for the detection of HCV in a simple, rapid, and cost-effective manner, which will be useful in resource-limited settings to allow the identification of individuals in need of HCV treatment.
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