Development and application of multiplex PCR of vibrios pathogenic to cultured large yellow croaker,Pseudnosciaena crocea (Richardson)

In recent years,Vibriosis was the most severe disease of cultured large yellow croaker(Pseudnosciaena crocea) in Zhejiang province,China,resulting in great economic losses. The clinical symptoms included anorexia,floating upward and swimming alone,pterygiophore hyperaemia,ulceration,ascites and lippitude. It was epidemic from June to October, with peak on July to August. The mortality rate varied from 30 % to 40 % ,even up to 80% sometimes. It costed only one week from some fish infected to almost all dead. Three bacterial pathogens accounted for the majority of large yellow croaker vibriosis in Zhejiang province,China. They were Vibrio alginolyticus,Vibrio parahaemolyticus and Vibrio harveyi. In this study,three special oligonucleotide primers for multiplex PCR amplification was designed,based on the diversity of the collagenase gene sequences between Vibrio alginolyticus and Vibrio parahaemolyticus,and the partial ToxR gene specific detection of Vibrio harveyi. The reaction conditions of the multiplex PCR were optimized and PCR products were sequenced. Specificity and sensitivity of multiplex PCR were studied. Finally,the multiplex PCR is well developed successfully allowing the detection of the three bacterial pathogens in one PCR tube with relatively equal intensities DNA bands when analyzed in an agarose gel. Positive control samples which contained DNA of Vibrio alginolyticus,Vibrio harveyi and Vibrio parahaemolyticus yielded evident amplification products showing the expected DNA fragments of 737 bp,382 bp and 271 bp respectively. The sensitivity of multiplex PCR was 102-103 CFU/mL. When it was applied to detect the vibrios in livers and kidneys of fish which were artificially infected,the vibrio was detected in five of six samples,which was as same as the vibrio formerly injected. The result was coherence with that of API 20E method;when it was applied to detect the vibrios in different tissue samples of large yellow croaker and water samples collected from fishery in epidemic season,one of 16 tissue samples produced positive bands of Vibrio harveyi,and seven of 16 water samples produced positive bands of one or two vibrio species. The coherence tested by multiplex PCR and API 20E methods was 93.75%. The results showed that the multiplex PCR could be used not only to diagnose the clinically diseased large yellow croaker, but also to recognize the carrier. What is more,there are vibrios pathogenic to cultured large yellow croaker in the aquatic environment. It can be concluded that the multiplex PCR is specific and sensitive. It is a reliable tool for identification of the major Vibrios pathogenic with less time and cost,and it can be used in quick diagnose and epidemiology investgation of vibriosis of cultured large yellow croaker.