Study On Rapid Detection Method Of Vibrio Harveyi

Vibrio harveyi is a vibrio bacterium,especially in shrimp.Once the shrimp infected with Vibrio harveyi can not be treated in time,the shrimp often have different degrees of congestion and ulceration on the surface muscles and some parts can illuminate in the dark,and will die after feeding for a period of time,bringing huge to the breeding industry.Therefore,it is important to establish methods for farmers and regulators to monitor Vibrio harveyi.At present,the detection methods of Vibrio harveyi mainly include three kinds of traditional medium culture methods,immunological analysis methods and molecular biological methods.The traditional culture method of Vibrio harveyi has problems such as long time-consuming and trivial operation.The existing immunological analysis method of Vibrio harveyi is mainly based on enzyme-linked immunosorbent assay and immuno-paper chromatography.The colloidal gold labeling chromatographic strip method is simple and easy to operate,and the reaction takes only ten minutes.It is suitable for on-site detection,but its sensitivity is low,mainly qualitative and semi-quantitative.Therefore,it is necessary to study the more sensitive and accurate quantitative detection method of Vibrio harveyi immunochromatography.In the molecular biology method,the reverse transcription loop-mediated isothermal amplification?LAMP?method and the RAA?Recombinase Aided Amplification?method are thermostatically amplified,and the pathogen can be amplified by using a simple constant temperature device.The method is simple and does not need to be used.Such a temperature changing device such as a PCR instrument greatly reduces the detection conditions.In order to develop a method suitable for on-site monitoring of Vibrio harveyi and laboratory to rapidly identify Vibrio harveyi,this paper first studied the method of detecting Vibrio harveyi based on magnetic immunochromatographic test strips,by optimizing antibody coupling.The preparation method of the magnetic immunochromatographic test strip of Vibrio harveyi was established by adding the ratio of the amount and the chromatographic solution.In order to improve the detection sensitivity of the test strip,the antibody coating concentration of the C and T lines was further optimized.Therefore,a preparation method of magnetic immunochromatographic test strip suitable for on-site detection is established,and the detection performances such as accuracy,sensitivity and specificity are evaluated.Secondly,based on the principle of molecular biology,through the optimization of detection time,primer screening and other detection processes,LAMP and RAA were established to detect Vibrio harveyi,and its accuracy,sensitivity and specificity were evaluated.The main results are as follows:1.The formaldehyde-inactivated Vibrio harveyi ATCC 33842 was used as the inactivated antigen to immunize New Zealand white rabbits to obtain a polyclonal antibody against Vibrio harveyi with a titer of more than 1:80000.The ELISA analysis found that the antibody was only Specific reaction to Vibrio harveyi.The self-made Vibrio cholerae polyclonal antibody was coupled to the surface of the magnetic nanomaterial,and the magnetic immunochromatographic test strip of Vibrio harveyi was constructed by the double-anti-sandwich method.By optimizing the amount of antibody coupled to the magnetic beads,the C-line and T-line coating antibody concentration,and the test strip chromatography system,the amount of 1 mg magnetic bead-conjugated antibody was 140?g,and the C-line and T-line concentrations were both 2 mg/mL.The content of Tween-20 is 10%?V/V?,the concentration of BSA is 0.2mg/m L,and the pH of BS buffer is 9.The test strip has the best detection effect,and the detection based on magnetic nanometer test strip is established.Method of Vibrio harveyi.The qualitative sensitivity of Vibrio harveyi magnetic immunochromatographic test strip is 1×10³ CFU/mL,and the quantitative sensitivity is 5×10² CFU/mL,which can be qualitatively and accurately quantified,and both detection limits are in Harvey.Within the limit of death of Vibrio,the sensitivity is increased by more than 100 times compared with the existing colloidal gold test strip,and the detection time is 10²0minutes.It is easy to operate and suitable for on-site detection.2.According to the design principle of LAMP primers,5 sets of different primers were synthesized.By optimizing the amplification temperature and primers,a method for detecting Vibrio harveyi at a constant temperature amplification of LAMP at 60°C was established.Through specific evaluation,it was found that the LAMP method constructed in this study only responded to Vibrio harveyi from different sources and showed good specificity.The detection limit of this method is 7.55 copies/?L,which is 10⁴ times higher than that of the traditional PCR method,and the detection time is 60minutes.The RAA primers and probes were designed independently,and the best primers were screened by the combination of different primers.The specificity and sensitivity of the established RAA detection method were evaluated.The method was found to have good specificity and the detection limit was 7.55×10^-1 copies/?L,the sensitivity is 10⁵ times higher than PCR,and the detection time is 20 minutes,which is a better method for rapid detection of Vibrio harveyi.The detection time is greatly shortened,and it is more suitable for rapid identification and screening of Vibrio harveyi in a conventional laboratory.In summary,this paper studies the rapid detection method of Vibrio harveyi on the basis of immunology and molecular biology,and establishes a magnetic immunochromatographic test strip suitable for on-site detection of Vibrio harveyi.The method established a LAMP and RAA method that is more suitable for routine identification and screening of Vibrio harveyi in conventional laboratories.All three detection methods shorten the detection time of Vibrio harveyi and achieve rapid and efficient detection.