title: Detecting a novel Eriocheir sinensis reovirus by reverse transcription loop-mediated isothermal amplification assay.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors: Wuxi Fisheries College, Nanjing Agricultural University, Nanjing, China.
authors: Y
authors: Y
authors: Ma
authors: Wuxi Fisheries College, Nanjing Agricultural University, Nanjing, China.
authors: T
authors: T
authors: Dai
authors: Wuxi Fisheries College, Nanjing Agricultural University, Nanjing, China.|Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China.
authors: A
authors: A
authors: Serwadda
authors: Wuxi Fisheries College, Nanjing Agricultural University, Nanjing, China. shenhs@ffrc.cn.|Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China. shenhs@ffrc.cn.
authors: H
authors: H
authors: Shen
mesh_terms: D000818:Animals; D017756:Aquaculture; D003386:Brachyura / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012087:Reoviridae / Q000235:genetics / Q000302:isolation & purification*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers
keywords: 
  Eriocheir sinensis
  ; RT-LAMP; RT-PCR; calcein; rapid detection; reovirus