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

UNLABELLED: 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 dNTPs, 4 mmol l⁻¹ MgSO₄, 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.