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Development of a Melting Curve-Based Triple Eva Green Real-Time PCR Assay for Simultaneous Detection of Three Shrimp
Xuan Dong1,2, Yujin Chen1,3, Haoyu Lou1
1State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Laboratory for Marine Fisheries Science and Food Production Processes, Laoshan Laboratory, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Qingdao 266071, China.
Abstract:
Infections with Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1) pose significant challenges to the shrimp industry. Here, a melting curve-based triple real-time PCR assay based on the fluorescent dye Eva Green was established for the simultaneous detection of EHP, IHHNV, and DIV1. The assay showed high specificity, sensitivity, and reproducibility. A total of 190 clinical samples from Shandong, Jiangsu, Sichuan, Guangdong, and Hainan provinces in China were evaluated by the triple Eva Green real-time PCR assay. The positive rates of EHP, IHHNV, and DIV1 were 10.5%, 18.9%, and 44.2%, respectively. The samples were also evaluated by TaqMan qPCR assays for EHP, DIV1, and IHHNV, and the concordance rate was 100%. This illustrated that the newly developed triple Eva Green real-time PCR assay can provide an accurate method for the simultaneous detection of three shrimp pathogens.
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