| Perch (Lateolabrax japonicus), commonly known as flower perch, seven stars bass, is a major marine fish species of sea-cage culture.According to incomplete statistics, in 2006 the number of cage culture sea bass in Zhejiang province was more than 30,000 boxes, and annual output was nearly 10 thousand tons, with several million dollars production value. But in recent years, with the size of cage culture expanding, perch-diseases have become increasingly prominent, and it has become a major bottleneck restricting of the development of aquaculture. A serious and infectious epidemic of Lateolabrax japonicus cultured in net-cage in Xiangshan,Ningbo,Zhejiang Province,broke out from May to June 2007. And in May 2008, the disease occered again in Xihu Harbor with a mortality of more than 30%. As the main object of disease-fish was about 2 ages above bass, it caused a great economic losses. Except for the representation of surface black, anorexia, weight loss, decreased activity,the main symptom of diseased fish was the appearance of many 1-2mm size white spots in the liver,spleen and kidney. The local farmers named it visceral white spot disease.In this study, use typical symptoms of the dying sick fish as a material, through pathogen isolation, artificial infections, pathogen morphology, physiological and biochemical characteristics and 16S rRNA, heat shock protein HSP60 sequence analysis and PCR,LAMP molecular detection technology, we made a research of the pathogen analysis and diagnostic technology with a view to provide reference for prevention and treatment of cage culture of sea bass- diseases.The study was carried out with two parts.First, pathogen identification. A Gram-negative, Curved short rod shaped bacterium designated as LY-1 was isolated from the infected fish.Which was about in the size of 1.4μm×0.8μm, sensitive to Vibrio inhibitors O/129 (150 ug / ml), positive in oxidase and catalase, with typical character of vibrio genus. its physiological and biochemical indices were most closest to the standard strains of Vibrio harveyi.Sequence alignment of 16SrDNA, HSP60 respectively and made evolutionary tree, it showed that LY-1 had a highest similarity with the Vibrio harveyi. In combination with other identification means, including morphological, physiological and biochemical indicators, the LY-1strain was identified as Vibrio harveyi.Second, establish rapid detection method of Vibrio harveyi. using vibrio ToxR gene to design primers and amplify part of the fragment sequencing of Vibrio harveyi LY-1. Design specific primers of PCR and LAMP amplification to successfully established two kinds of special PCR and LAMP rapid detection method of Vibrio harveyi. The established PCR technology could detect a minimum detection limit was 1pg of Vibrio harveyi DNA. PCR amplification of Vibrio harveyi LY-1 and 8 strains of Vibrio standard strains ,we found that except LY-1 and the standard strains of Vibrio harveyi could amplify 393 bp specific fragment, the amplification results of other Vibrio were negative.In the experiment, we adopted two-point temperature PCR amplification. From DNA extracted to PCR end, the whole process needed just two hours, greatly reducing the detection time.LAMP results analysis shows that, LAMP method reaction time was short, it just needed a 65℃conditions to incubate for 45min to complete the amplification. specificient to Vibrio harveyi. And the minimum detectable amount was 1fg, which was three magnitude sensitivity higher than PCR method.This paper reported Vibrio harveyi as the pathogeny of bass visceral white spot disease. The two rapid detection method of Vibrio harveyi method had the virtue of simple operation,short detection time and high sensitivity. It provided a reference for Vibrio harveyi-disease. Using LAMP amplication to detect Vibrio harveyi is the first report in China.
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