title: Rapid detection of mud crab dicistrovirus-1 using loop-mediated isothermal amplification.
abstract: 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 μmol/L F3/B3, 1.6 μmol/L FIP/BIP, 6 mmol/L Mg(2+), 0.8 mmol/L dNTPs, and 0.8 mol/L betaine, and completed in 1h at 62°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.
authors.affiliation: Key Laboratory of Aquatic Product Processing, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China. Electronic address: guozhixun1@163.com.
authors.forename: Zhixun
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Key Laboratory of Aquatic Product Processing, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China; College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China.
authors.forename: Di
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Aquatic Product Processing, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China.
authors.forename: Hongling
authors.initials: H
authors.lastname: Ma
authors.affiliation: Key Laboratory of Aquatic Product Processing, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China.
authors.forename: Youlu
authors.initials: Y
authors.lastname: Su
authors.affiliation: Key Laboratory of Aquatic Product Processing, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Feng
authors.affiliation: Key Laboratory of Aquatic Product Processing, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China.
authors.forename: Liwen
authors.initials: L
authors.lastname: Xu
mesh_terms: D000818:Animals; D003386:Brachyura / Q000821:virology*; D002681:China; D017931:DNA Primers / Q000235:genetics; D056629:Dicistroviridae / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013696:Temperature; D013997:Time Factors; D015678:Viral Structural Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D015678:Viral Structural Proteins
keywords: Loop-mediated isothermal amplification; Mud crab dicistrovirus-1; Scylla paramamosain