| In recent years, turbot, with the advantage of fast growing, tasty meat, high value of goods, become the most popular farmed species in Liaoning coastal areas. The Huludao city had formed the iconic turbot breeding industry base, its turbot aquaculture area of 1.5 million square meters, the output value of 1.5 billion yuan, aquaculture production was 25,000 tons, accounting for about 58% of farmed turbot production in the country. However, with the rapid expansion of the scale of farming, industrial development speed significantly beyond disease control rate, according to the data of Fisheries Extension Center in Liaoning province, in recent years, Liaoning Provincial factory farming turbot had serious losses due to the disease, the average loss amount was up to 3 million per month, accounting for 98% of the total economic loss of all aquatic species.In the process of breeding, low water temperature will decrease the food intake of turbot, weak its system, then cause the bacterial disease outbreak, such disease is difficult to control once the outbreak, is bound to bring immeasurable loss to farmers. Due to the research about common pathogenic bacteria in the turbot breeding process in Liaoning provincial sea area relatively scarce and combined with the related rapid detection methods of pathogenic microorganism is still a blank, which limited the development of turbot aquaculture in Liaoning province.In order to solve the problem above, we did the systematic investigation on pathogenic microorganisms which can induce the turbot disease in Liaoning provincial sea area, and according to the results of the investigation, a rapid detection method for important pathogens associated was established, the main method was PCR detection, the concrete content includes the design of primers, and the validation of its specificity and sensitivity.The research did sample collection in the major breeding base of turbot in Liaoning province, and treated the lesion with the traditional methods of separation, purification, identification of the flora and the analysis of the 16s rRNA macro genome sequencing. The results show that the advantage bacterium group in the lesion of turbot was mainly Vibrio and Aeromonas, of which Vibrio alginolyticus, Vibro Splendidus, Aeromonas hydrophila and Aeromonas salmonicida were closely associated with turbot disease, threaten the healthy development of the turbot aquaculture industry. In view of the field study of Vibrio was more perfect and the study of Aeromonas are relatively scarce, this paper by Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas schubertii and Aeromonas sobria as the main research object, designed 12 pairs of specific primers, and verify the specificity and sensitivity of each primer. The result showed that the primer H-2, Sa-1, Sa-2, Sa-3, So-1, So-2, So-3, Sc-1 had strong specificity and sensitivity, can be used for the establishment of a single pathogen strains rapid detection method. At the same time, using primers H-2, So-3 and Sc-1 to establish simultaneous detection method for 3 pathogenic strains by multiplex primer PCR detection method, the detection method proven high specificity and sensitivityThis study provide necessary theoretical basis and improved method system for the detection and analysis of high pathogenic bacteria in the process of turbot breeding, which not only made a contribution for the future healthy development of turbot culture industry, at the same time provided a guidance for detecting other pathogenic microorganisms in aquatic product, and laid a foundation for promoting the healthy development of the aquatic products breeding industry in China. |
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