Molecular detection of genotype II grass carp reovirus based on nucleic acid sequence-based amplification combined with enzyme-linked immunosorbent assay (NASBA-ELISA).
Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons. The amplicons were hybridized to specific biotinylated DNA probes and the products were detected colorimetrically using horseradish peroxidase and a microplate reader. The new method is able to detect GCRV at 14 copies/μL within 5h and had a diagnostic sensitivity and a specificity of 100% when GCRV-II and non-target virus were tested. This NASBA-ELISA was evaluated using a panel of clinical samples (n=103) to demonstrate that it is a rapid, effective and sensitive method for GCRV detection in grass carp aquaculture.
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China.
Weiwei
W
Zeng
Liaoning Animal Medical Research Institute, Shenyang, 110164, China.
Wei
W
Yao
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China.
Yingying
Y
Wang
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China.
Yingying
Y
Li
FLI Insel Riems, Sudufer 10, 17493 Greifswald-Insel Riems, Germany.
Sven M
SM
Bermann
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China.
Yan
Y
Ren
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China.
Cunbin
C
Shi
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China.
Xinjian
X
Song
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China.
Qiwen
Q
Huang
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China.
Shuchen
S
Zheng
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong China. Electronic address: sunny_929@163.com.
Qing
Q
Wang
D000818:Animals; D002347:Carps; D002681:China; D003124:Colorimetry / Q000379:methods*; D003955:Diagnostic Tests, Routine / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D005838:Genotype; D012087:Reoviridae / Q000235:genetics / Q000302:isolation & purification*; D012088:Reoviridae Infections / Q000662:veterinary* / Q000821:virology; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
GCRV II; Grass carp reovirus; NASBA-ELISA