Establishment Of Rapid Detection Methods For Three Kinds Of Bacteria In Brachymystax Lenok

The Brachymystax lenok belongs to the genus Salicidae,and the distribution of wild squid in the genus Diptera has been gradually reduced in recent years.The wild population has declined sharply.In order to prevent the extinction of this population,the protection and domestication of the squid are particularly important.In recent years,in the domestication and breeding of the squid,it is found that the squid will have anorexia,living alone,water rotation,surface ulcer and liver and kidney bleeding.Symptoms,three bacteria are isolated from the liver,kidney,intestinal fluid and sputum of the diseased fish,which are Aeromonas sobria（AS）,Edward Edwards tarda（Et）and Aeromonas hydrophila（AH）.The three kinds of bacteria are conditional pathogens.In the artificial domestication of squid,there is an urgent need for a quick and simple method for accurate and rapid diagnosis of pathogenic bacteria for early prevention and treatment of diseases.OBJECTIVE:To establish a method for rapid detection of three kinds of bacteria,and to detect these three kinds of bacteria which infected the Brachymystax lenok in time to prevent the outbreak and large-scale spread of the squid,and to provide protection for the domestication of Brachymystax lenok.METHODS:In this experiment,three strains were isolated and purified from the liver,kidney,intestinal effusion or silk of the diseased fish.Using four identification media,the strain type was initially determined according to the morphological characteristics on the medium,and used more than 20antibiotics for drug susceptibility experiments,and 16S rRNA was used to sequence the isolated and purified bacteria.Rapid detection of Aeromonas hydrophila,Edwardsiella tarda and Aeromonas sobria was established by loop-mediated isothermal amplification（LAMP）.A specific LAMP primer was designed for the zipA gene of the Aeromonas sobria,pilin gene of the Aeromonas hydrophila,and the ethA gene of Edwardsiella tarda.Five primers were designed for each strain.The reaction system is 25μL based on the following content,where in the concentration of the loop primer is 40 pmol,the concentration of the inner primer is 40 pmol,the reaction concentration of the outer primer is 5 pmol,and the concentration of the buffer is 2.5 mol/L.The concentration of magnesium ion is 150 mmol/L,the concentration of dNTP is 10mmol/L,the concentration of polymerase is 8 U,the template is 2μL,and deionized water is added to 25μL.The temperature gradient is set between 61-65°C.The system was placed in a water bath with various temperature gradients to compare the optimal reaction temperature,and then LAMP amplification was carried out under constant temperature conditions to optimize the optimal reaction conditions,including optimization of magnesium ion,buffer,and primer ratio.And the amount of dNTP,the amplification results were compared and determined by agarose electrophoresis and nucleic acid dye color method in the optimization.After 10 times dilution of DNA from 10^-1111 to 10^-22 as a template,LAMP reaction to detect its sensitivity;extraction of Aeromonas salraonicida、Aeromonas caviae、Aeromonas media DNA As a template,the reaction was carried out using the above optimized system,and the results were observed,whether there was white precipitate,whether there was color change after the dye was added,and whether there was a trapezoidal band to indicate the specificity of the established method;The established LAMP method is compared with the traditional identification method and real-time PCR to ensure that the established LAMP method is accurate,does not have high false positives,and can quickly and efficiently detect the three pathogenic bacteria.RESULTS:A set of best primers was selected for each bacterium.The optimal LAMP reaction system was that the concentration of the loop primer was 40 pmol,the inner primer was 30 pmol,the outer primer was 5 pmol,and the buffer was 2.5 mol/L.The concentration of magnesium ion is 150 mmol/L,of dNTP is10 mmol/L,of polymerase is 8 U,and the template is 2μL The result can be observed in 60 min.and the minimum detection concentrations of Aeromonas hydrophila,Aeromonas sobria and Edwardsiella tarda are6.35×10^-10,1.663×10^-9,9.86×10^-1010 ng/μL.The method has good specificity and high sensitivity.The detection time of the source can be controlled within 1 day.The traditional detection method takes at least two days,while real-time PCR can amplify genomic DNA at a concentration of 10^-1111 ng/μL,but the required loading environment also has high requirements,which is not suitable for popularization in clinical and production.Conclusion:The method requires simple,accurate and efficient instruments,convenient operation,and is suitable for popularization in clinical and production.