Development of a reverse transcription‐loop‐mediated isothermal amplification ( RT ‐ LAMP ) system for rapid detection of HDV genotype 1

The object of this study was to develop a reverse transcription‐loop‐mediated isothermal amplification ( RT‐LAMP ) assay for detecting hepatitis D virus ( HDV ) genotype 1. With an alignment analysis, a highly conserved sequence (nt 820–1020) was chosen as a suitable target to design LAMP primers. The optimal condition of RT‐LAMP was a 25‐μl reaction volume, which consists of the following components: 1·6 μmol l −1 each of FIP and BIP, 0·2 μmol l −1 each of F3 and B3, 1·5 μmol l −1 dNTP s, 4 mmol l −1 MgSO 4 , 8 U Bst DNA polymerase, 2U M‐MLV and 2 μl extracted RNA sample. The amplification reaction was carried out at 65°C for 50 min. Compared with conventional qualitative or quantitative real‐time reverse transcription polymerase chain reaction, the results of RT‐LAMP indicated a 1000‐fold increase in sensitivity for detecting HDV. There was no cross‐reaction for the RT‐LAMP method between HDV 1 and HIV, HAV, HBV, HCV and HEV. Significance and Impact of the Study The results indicate that RT‐LAMP is a simple, rapid, specific, highly sensitive and cost‐effective, field‐based method for detecting HDV 1. The RT‐LAMP assay is an acceptable alternative to diagnose the HDV genotype 1 and to investigate its epidemiology for clinical laboratories lacking specialized equipments.