title: 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/Mn2+-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/Mn2+-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.
authors.affiliation: The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, Hebei Province, China.|The Experiment Center of Pathogen Biology, Chengde Medical University, Chengde, Hebei Province, China.
authors.forename: Na
authors.initials: N
authors.lastname: Zhao
authors.affiliation: The Experiment Center of Pathogen Biology, Chengde Medical University, Chengde, Hebei Province, China.
authors.forename: Jinxia
authors.initials: J
authors.lastname: Liu
authors.affiliation: The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang, Hebei Province, China.
authors.forename: Dianxing
authors.initials: D
authors.lastname: Sun
mesh_terms: D002681:China; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: genotype 1b; genotype 2a; genotype identification; hepatitis C virus; loop-mediated isothermal amplification; reverse transcription