Establishment and Application of Amplicon Rescue Multiplex PCR(Arm-PCR) for the Simultaneous Detection of Five Major Bacterial Pathogens from Marine Aquaculture Animals
Abstract：Vibrio anguillarum, V. harveyi, Aeromonas hydrophila, Edwardsiella tarda and V. parahaemolyticus are five major bacterial pathogens isolated from marine aquaculture animals in China. In this report, five set of nested specific primers were designed based on the pathogenic factor genes and a specific amplicon rescue multiplex PCR (Arm-PCR) for the simultaneous detection of these five major bacterial pathogens were established. The concentration of primer Mix, Mg2+, dNTPs, Taq DNA polymerase and the annealing temperature in the first step of Arm-PCR were optimized in this study. With summarizing the results of the experiment, in 50 μL of reaction volume, the optimized parameters were 10×PCR Buffer(20 mmol/L Mg2+) 5 μL, dNTPs(2.5 mmol/L each) 5 μL, Taq DNA polymerase(2.5 U/μL) 0.6 μL, 10×primer Mix(2 μmol/L) 5 μL, each template 1 μL. The annealing temperature was 55℃. The result showed that the Arm-PCR reported here could produce specific amplicons in one tube simultaneously. The expected sizes were 144, 190, 266, 315 and 371 bp for V. anguillarum, V. harveyi, A. hydrophila, E. tarda and V. parahaemolyticus, respectively. The sensitivities of the Arm-PCR were 1.745, 1.847, 16.000, 28.126 and 369.900 pg to above bacterial genomic DNA. There were no cross reactions with genomic DNA from healthy fish and other common bacteria, such as Escherichia coli, V. alginolyticus, Pseudoalteromonas tetraodonis, Bacillus subtilis, accorrding to the Arm-PCR. In 2012, the established Arm-PCR method was applied to the detection of 24 bacterial isolates from diseased fish and five strains of E. tarda, three strains of A. hydrophila, two strains V. harveyi and two strains V. parahaemolyticus were identified. The results showed that the method was reliable and practicable. The Arm-PCR method reported here can be used not only for the simultaneous specific detection and epidemiological survey of above five pathogens in marine aquacultural animals, but aslo for the development of microarray detection methods in the future.
