Establishment of a loop-mediated isothermal amplification assay for de-tection of carp edema virus
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1. National Demonstration Center for Experimental Fisheries Science Education(Shanghai Ocean University), Shang-hai 201306, China;
2. Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China

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S941

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    Abstract:

    Common carp (), the earliest farmed fish in the world, is one of the most important and economically significant species in freshwater aquaculture in China. In recent years, carp edema virus (CEV) disease, a newly emerged infectious disease in common carp, has been discovered in many countries, threatening the healthy development of common carp farming. Due to a lack of CEV-sensitive cell lines, detection of CEV relies on methods that use electron microscopy and polymerase chain reaction (PCR); however, these methods are not used universally for clinical diagnosis because they require expensive equipment and a long response time. Therefore, there is an urgent need to establish a simple, accurate, and rapid diagnostic method to detect CEV. Loop-mediated isothermal amplification (LAMP) is a novel amplification technique used to produce DNA copies within 1 h. Furthermore, the results can be judged based on turbidity or by the addition of SYBR Green I to the reaction products and observation of color change within the reaction system. LAMP technology has played an important role in human medicine, animal medicine, and food safety, and has gradually been developed for industrialization and commercialization. In this study, two pairs of specific primers were designed based on the conserved sequence of the gene encoding the CEV nucleus protein, , and a recombinant plasmid was constructed for use as a standard template for amplification. Then, a LAMP detection method for CEV was established after optimizing the reaction conditions. The optimal LAMP reaction system for CEV detection included F3/B3 0.2 μmol/L, FIP/BIP 1.2 μmol/L, betaine 0.7 mol/L, Mg2+ 8.0 mmol/L, and dNTPs 1.2 mmol/L and was performed at 62℃ for 60 min. The amplified products included a ladder band on agarose electrophoresis and green products, which were observed directly by the addition of SYBR Green I. The sensitivity of the detection method for CEV was 10 copies/μL, which is 100-fold higher than the traditional PCR method. Moreover, CEV-LAMP was specific for detecting CEV without cross reaction with other viral genomes, including spring viremia of carp virus, koi herpesvirus, cyprinid herpesvirus 2, and infection spleen and kidney necrosis virus. This method has been used for the rapid detection of CEV in diseased koi carp. The results were accurate and reliable, providing an effective and useful method for the on-site diagnosis of CEV.

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翟立文,刘文枝,许晨,范玉顶,江南,周勇,曾令兵. 鲤浮肿病病毒环介导等温扩增(LAMP)检测方法的建立[J]. Jounal of Fishery Sciences of China, 2019,[volume_no](5):1004-1013

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  • Online: September 18,2019
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