Establishment Of Detection Methods For Pathogenic Vibrio By Liquid Phase Chip And LAMP

Vibrio was widely distributed in the oceans,rivers and lakes,from the poles to the equator of the extreme cold water in a tropical environment were distributed,with the improvement of living standards,the growing demands for seafood was much bigger than before,Vibrio had already become an important pathogen threat to aquaculture and human health,but there were many kinds of Vibrio,which had been a huge challenge in terms of detection and control,so the establishment of multiple detection and detection of Vibrio base was very necessary.Vibrio parahaemolyticus was an important pathogenic Viblrio in aquaculture threat,leading to the enormous economic losses each year,Vibrio parahaemolyticus had two chromosomes,each chromosome had a different gene encoding outer membrane protein ompA,respectively VP0764 and VPA1186,which were designed to take these two pairs of F/R primers,respectively with BamH and Sacl restriction sites,with BL21 vector for protein expression,purification and identification to prepare the polyclonal antibody,and preparation of immunomagnetic cell capture.The virulence gene(vptdh)of Vibirio parahaemolyticus and specific gene(vptox),enterotoxin base of Vibrio cholerae(vcctx)and outer membrane protein gene(vcomp)and hemolysin gene(vchly),hemolysin gene of Vibrio mimicus(vmha)and specific gene(vmtox),hemolysin gene of Vibrio vulnificus(vvhA)and Vibrio vulnificus multiple toxin gene(vvrtx),collagen enzyme gene of Vibrio alginolyticus(vacol)gene were designed as target gene and specific microspheres for the experimental study of the technique of liquid phase chip.PCR amplification was carried out on five kinds of Vibrio cholerae,Escherichia coli and Salmonella,reading the hybridization with specific probes product,it turned out that liquid chip technology had good specificity;different concentrations of PCR products hybridized with specific probes,proving its sensitivity was high;ten specific probes respectively were hybridized with both single PCR products and different concentrations of PCR products,confirming the operability of liquid chip technology for multiple testing.Vibirio vulnificus hemolysin A gene(hemolysin gene A HA)and multiple toxin(repeats in toxin,RTX)were researched virulence genes of two genes were designed,primers for LAMP detection of target gene and artificial samples,judging the detection results through visual observation of white precipitate,further confirming the inspection results.By agarose gel electrophoresis,two gene detection sensitivity was 10 CFU/ml,simulated the sensitivity of sample detection were 1×102 CFU/g(VV-RTX)and 1×104 CFU/g(VV-HA).Comparing with ordinary PCR,LAMP had much higher sensitivity,shorter time,lower cost,which was moer suitable for the base layer.