Development of hepatitis C virus by fluorescent real-time reverse transcription loop-mediated isothermal amplification method

Abstract

Objective To establish a rapid, sensitive, and specific quantitative method to detect hepatitis C virus. Methods A primer set targeting HCV 5'UTR was designed. The isothermal amplification was performed by the Bst DNA polymerase and AMV reverse transcriptase, under the temperature of 60℃ for 60 min. The signal was monitored by SYBR Green Ⅰ. Results One hundred and twenty positive serum samples, confirmed by the real-time PCR. All were detected by the isothermal amplification, while 110 healthy subjects' samples were negative by the both methods. The lower detect limit was determined to 10 IU/ml HCV-RNA, by the assay on serial dilutions of the quality control standards obtained from clinical investigation center of MOH. Conclusion A real time reverse loop-mediated isothermal amplification method was developed to detect HCV, with the characteristic of rapidity, high sensitivity and specificity. Key words: Fluorescence quantitation; Reverse loop-mediated isothermal amplification; Hepatitis C virus; Real-time PCR