Rapid detection of mud crab dicistrovirus-1 using loop-mediated isothermal amplification

摘要

Mud crab dicistrovirus-1 (MCDV-1) was isolated from the mud crab (Scylla paramamosain), resulting in mass mortality and widespread economic loss in China. In this study, a detection method for MCDV-1 using loop-mediated isothermal amplification was developed. Two pairs of primers targeting the VP2 gene were designed. These primers were the outer primers F3 and B3, and the inner primers FIP and BIP. Optimal amplification was carried out using 0.2 mu mol/L F3/B3, 1.6 mu mol/L FIP/BIP, 6 mmol/L Mg2+, 0.8 mmol/L dNTPs, and 0.8 mol/L betaine, and completed in 1 h at 62 degrees C. The products demonstrated a ladder pattern on agarose gel electrophoresis and could also be detected visually according to turbidity, or by adding SYBR Green I and observing a color change from orange to green. The proposed method could specifically amplify MCDV-1 gene fragments. Sensitivity assay revealed that six copies of the viral genome could be detected by this method, which was 1000-fold more sensitive than that of conventional PCR using constructed plasmid as amplification template. At clinical sample level, sensitivity of LAMP was 100-fold higher than that of conventional PCR. (C) 2014 Elsevier B.V. All rights reserved.