Detection of HCV genotypes 1b and 2a by a reverse transcription loop‐mediated isothermal amplification assay

Abstract

Hepatitis C virus (HCV) genotypes 1b and 2a are the major cause of liver disease in northern China; however, conventional detection tools are labor-consuming, technically demanding, and costly. Here, we assessed the specificity, sensitivity, and clinical utility of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of HCV genotypes 1b and 2a. Firstly, clinical samples were collected from HCV genotype 1b and 2a infected patients and the RNA were extracted. Secondly, specificity of RT-LAMP assay for detection HCV genotypes 1b and 2a were tested against viral genomes of other hepatitis viruses. Sensitivity of RT-LAMP assay was determined using serial dilutions of standard HCV genotypes 1b and 2a. The amplified products were detected by both electrophoresis and calcein/Mn -dependent visual methods. Finally, we compared the clinical detection rate of RT-LAMP to that of real-time PCR. RT-LAMP assay showed high specificity to detect HCV genotypes 1b and 2b since there was no cross-reactivity with other hepatitis viruses. Sensitivity of RT-LAMP was 100 IU/mL for both genotypes detected by either electrophoresis or calcein/Mn -dependent visual methods. The detection rate of RT-LAMP assay in clinical samples was also comparable to that of real-time PCR without significant difference between the both assays. This study proposes a newly developed RT-LAMP assay for detection of HCV genotypes 1b and 2a. RT-LAMP is highly specific, sensitive, and simple diagnostic tool which would be useful for screening and early diagnosis of HCV especially in resource-limited environments.

Colorimetric LAMP microfluidic chip for detecting three allergens: peanut, sesame and soybean

“…In recent years, LAMP, a nucleic acid detection technology, has been established by Notomi et al . (2000) for detecting and identifying viruses, bacteria, animals and plants 21 – 23 . Under Bst DNA polymerase, they designed four specific primers to trigger the isothermal amplification (60–65 °C) of the target DNA within 60 min.…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…Positive LAMP reaction was indicated with green colour, whereas negative LAMP reaction indicated with orange colour. To name a few, calcein has been used in detection of Trypanosoma cruzi (Besuschio et al 2017), avian influenza H5 virus (Liu et al 2013), hepatitis C virus (Zhao et al 2017), human papillomavirus , human coronavirus (Geng et al 2016) and Flavobacterium columnare in tilapia fish (Suebsing et al 2015). Calcein-based end-point detection is most commonly used due to its high sensitivity and notable colour change for naked eye observation (Fischbach et al 2015).…”

“…RT-LAMP was established for Dengue virus (Hu et al 2015;Lau et al 2015), influenza viruses (Nakauchi et al 2014;Bao et al 2015;Kim et al 2016a), Hepatitis C (Nyan and Swinson 2016;Zhao et al 2017), Ebola virus (Oloniniyi et al 2017), respiratory syncytial virus (Hoos et al 2017), zika virus (Tian et al 2016;Wang et al 2016;Chotiwan et al 2017) and Middle East Respiratory Syndrome coronavirus (Mers-CoV) Lee et al 2016). Apart from helping in diagnosis, the RT-LAMP can also be used as an epidemiologic surveillance system for human virus infections.…”

Rapid visual detection of hepatitis C virus using a reverse transcription loop-mediated isothermal amplification assay

“…Currently, several LAMP-based assays have been designed for the detection of various pathogens including HCV, but they detect a limited number of genotypes and a small number of clinical specimens have been used for assay validation (Kargar et al, 2012;Nyan and Swinson, 2016;Wang et al, 2011;Yang et al, 2011;Zhao et al, 2017). In this study, we introduce an RT-LAMP assay for the detection of HCV in Thailand, in which the reaction color is observed in a single closed tube.…”