Rapid and simple detection of Hepatitis C virus by reverse transcriptase -loop- mediated isothermal amplification method

Abstract

Hepatitis C virus (HCV) infection frequently causes chronic hepatitis, which progresses to liver cirrhosis and hepatocellular carcinoma. HCV infection has now become a serious health problem because at least 170 million people worldwide are currently infected with HCV. Molecular diagnostics are revolutionizing the clinical practice of infectious disease. Loop-mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification. This simple and rapid technique relies on strand-displacing DNA synthesis performed using the Bst DNA polymerase large fragment under isothermal conditions in the temperature range of 60–65°C, thereby obviating the need for a thermal cycler. The method has also been rendered applicable to RNA genomes by combining it with reverse transcription reactions (RT-LAMP). RT-LAMP is characterized by the use of 6 different primers. For 110 quantities sera, Nested RT-PCR and RT-LAMP were done. At the end of theLAMP reaction, SYBR Green was used for identifying negative and positive results. The PCR sensitivity up to 80 particles was observed and the LAMP sensitivity test was verified up to 8 particles. The data presented in this study suggested that the RT-LAMP assay is more sensitive than RT-PCR by picking up 5 additional cases that were negative by RT-PCR. These findings demonstrate that detection of HCV using RT-LAMP was more sensitive than the Nested RT-PCR. The RT-LAMP assay developed in this study is simple, rapid, and cost effective as well as highly sensitive and specific. Key words: Hepatitis C virus, RT-LAMP, nested RT-PCR.