Technical Development Of Detection Techniques For Common Pathogens Of Aeromonas In Cultured Frogs

The industry of frog breeding can meet our needs of eating,medication,etc.However,the outbreak of some diseases often causes great losses to frog farmers.Most of diseases are bacterial diseases during frog breeding.The traditional research methods are investigating the pathogen firstly,and then isolating and purifying the bacteria from the diseased frogs.After that,using a series of physiological,biochemical and molecular methods to identify the bacteria.Then,the pathogen is verified by the recurrent infection experiment.Finally putting up some suggestions to the selection of the therapeutic drug by antimicrobial susceptibility test.However,the process tends to be a long cycle,and many frogs have been illed seriously.In this paper,we chosed A.hydrophila?A.sobria?A.caviae and A.veronii to be the research focus which is the most common pathogens of Aeromonas for frogs.We establish the technical development of detection techniques for common pathogens of Aeromonas in cultured frogs by using environmental DNA technology and multiplex DNA amplification,and try to find some kinds of antibacterial drugs which have good antibacterial effects against the pathogens of the same genus,to provide new ideas and theoretical basis for the early prevention and control work of common bacterial diseases in the frog breeding.The main results and conclusions are as follows:1.Established an efficient and practical method for extracting environmental DNA from water sample.Using the filter membrane or precipitation method to extract environmental DNA from two different sizes of water body and conducting PCR amplification to detect the quality and quantity of environmental DNA.The method can be applied to different conditions of water sample.2.Two pairs of specific primers have been selected for PCR amplification of common pathogenic bacteria of Aeromonas by comparing the 16 S rDNA sequences of four common pathogens of Aeromonas and 14 other non-Aeromonas frog pathogens.The PCR amplification products are 229 bp and 688 bp,respectively.The primers can also be used to detect the common pathogenic bacteria of Aeromonas genus in water by combining environmental DNA technology.3.By comparing the gyrB gene sequences of four common pathogens of Aeromonas,such as A.hydrophila?A.sobria?A.caviae and A.veronii,four pairs of specific primers are designed and screened to identify these four pathogens of Aeromonas.The primers for identification of A.hydrophila has a PCR amplification product size of 266 bp;the primers for identification of A.sobria has a PCR amplification product size of 99 bp;the primers for identification of A.caviae has a PCR amplification product size of 518 bp;the primers for identification of A.veronii has a PCR amplification product size of 120 bp.Then we establish two Duplex PCR,one of them can take rapid test of A.hydrophila and A.veronii,the other can take rapid test of A.sobria and A.caviae.We can combine them to identify the four common pathogens of Aeromonas.4.Ten kinds of antibacterial drugs are selected according to the literature,which are used to take antimicrobial susceptibility test of A.hydrophila?A.sobria?A.caviae and A.veronii.The results shows that the kinds of antibacterial drugs have good antibacterial effects against the common pathogens of Aeromonas for frogs.