The novel Eriocheir sinensis reovirus (EsRV) is a pathogen that causes severe disease and high mortality rates in cultivated crabs. Here, we established a highly sensitive and specific rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that was cheaper and more suitable for field applications in crab aquaculture than those of traditional reverse transcription-polymerase chain reaction (RT-PCR) analysis. The amplification was completed within 45 min under isothermal conditions at 65°C. The RT-LAMP test for EsRV had a detection limit of 15 pg, and sensitivity was 100 times greater than that of conventional RT-PCR. The LAMP primers for EsRV were not amplified by other pathogen strains, indicating good specificity. In addition to detection by electrophoresis, RT-LAMP results were detectable by visual observations of reaction tube turbidity, and calcein was added to visually detect the amplification products. These results indicate that this highly convenient, rapid and sensitive RT-LAMP assay can be used to detect EsRV-infected aquatic organisms.
Significance and impact of the study: Tremor disease (TD) is one of the most serious diseases of Eriocheir sinensis. A novel E. sinensis reovirus (EsRV) was identified from E. sinensis afflicted with TD and caused high mortality. We developed a reverse transcription loop-mediated isothermal amplification assay with high specificity, sensitivity and rapidity to detect EsRV, which can be used to diagnose aquatic animal diseases, particularly where expensive diagnostic instruments are not available.
Keywords: Eriocheir sinensis; RT-LAMP; RT-PCR; calcein; rapid detection; reovirus.
© 2016 The Society for Applied Microbiology.