Different Expression Level Of Virulence Genes Of Vibrio Parahaemolyticus Under Different Environmental Samples And Establishing A Quantification Method Of Viable Vibrio Parahaemolyticus From Seafood

Vibrio parahaemolyticus is a gram-negative marine bacterium that is a major pathogen offood-borne gastroenteritis associated with consumption of raw or undercooked seafood, especiallyoysters, and it is widely present in estuarine, marine, and coastal environments throughout the world.In recent years, the infection cases of Vibrio parahaemolyticus grow sustainably, and bring a lot ofpotential safety hazard to aquaculture and seafood consumption. Studies have shown that majorvirulence factors of Vibrio parahaemolyticus is the hemolysin, thermostable direct hemolysin (TDH),tdh-related haemolysin(TRH), themolabile hemolysin(TLH). In addition, the infection rate of Vibrioparahaemolyticusin shrimp is90%. This is a big menace to the human health.Gene expression analysis has become increasingly important in the field of life science,and the qRT-PCR technology has recently reached a level of sensitivity, accuracy and practicalsimplicity allowing its use as a routine bioinstrumentation for pathogen detection, single nucleotidepolymorphism and gene expression analysis. The research about virulence gene expression of Vibrioparahaemolyticus has been started until now. But studies on virulence factors and virulencemechanisms of Vibrio parahaemolyticus reported in literature thus far are usually performed innutrient-rich synthetic growth media. It is of utmost importance to study virulence factor expressionunder conditions that are as close as possible to those the bacteria experience in the real world, inorder to understand the real mechanisms behind virulence gene expression. In addition, it isnecessary for setting an integral evaluation system of gene expression to select a stable gene as thereference gene. Currently there are lots of reports about screening reference gene for gene expressionresearch, while most of them were restricted to plants and animals, only few of them focused onpathogen. So this research was based on the water environment which contains Vibrioparahaemolyticus, and studied on Vibrio parahaemolyticus in shrimp samples, seawater samples,and filtered seawater samples and pure cultured, screened out a stable house-keeping gene asreference gene for different experiment materials and treatments. Then we used qRT-PCR, analyzedthe expression difference of tdh, trh and tlh gene of different strains of Vibrio parahaemolyticus which under different environmental conditions, tried to make contribution to research about themechanism of environmental factors control the virulence gene expression and the suitability ofdifferent virulence gene.Recently, with the development of molecular detection technologies, many detectionmethods of Vibrio parahaemolyticus from the seafood based on real-time PCR have been developed.Real-time PCR is faster and more sensitive than conventional PCR, so it has been widely used in thedetection of Vibrio parahaemolyticus. However, many new real-time PCR methods were based onDNA. The major limitation of the DNA-based qPCR method is the potential detection of both liveand dead, or non-culturable cells method is the potential detection of both live and dead, ornon-culturable cells. It would make us overestimates the risk of samples. The followed qRT-PCRtechnology, which uses mRNA as template instead of DNA, could decrease the ratio of false positivein detection for viable organism; get more accurate and more dependable result. So this research hasbeen established a quantification method of viable Vibrio parahaemolyticus based on RNA technique.The experiment result indicated:1. There is no efficient method to extract microorganism total RNA from food samples. It is a bigproblem for the following gene expression research. So first we optimized the Vibrioparahaemolyticus RNA extraction conditions from shrimp sample. We selected two differentbacterial cell recovery solutions,0.1%peptone water and0.85%physiological saline. There are twooptimized the method which could extract high quality RNA, which use manual homogenate and0.85%physiological saline, the extraction could be used in the subsequent gene expression analysisexperiment.2. Used the geNorm software to calculate and analyze the expression stability of fourhouse-keeping genes of Vibrio parahaemolyticus quoted from related researches. The expressionstability of the four reference genes ranked pvuA (2.906)> pvsA (3.197)> GAPDH (3.746)>rpoS(6.512). Further analysis revealed that the highest stability was observed in pvuA and pvsA.found the sequence of them was pvuA, pvsA, GAPDH and rpoS, after best reference gene numberevaluation, found using the geometric average of pvuA and pvsA as reference could regulate thetarget gene expression more exactly. So the pvuA and pvsA could be used as reference genes tostudy the virulence gene expression of Vibrio parahaemolyticus.3. Used qRT-PCR to analyze the expression of five strains bacteria virulence gene in differenttemperature seawater, filtered seawater and shrimp sample. the result indicated that the virulencegene from different bacterial strain shows different adaptive faculty to different environmentalconditions, such as trh and tlh express at high level when bacteria grown at low temperature.Secondly, the temperature dependency of tdh gene expression was not significant, but the tdh genefrom QD strains showed a higher starvation tolerance. And there is a big gap between the expressionlevel of strains which contain tdh and trh and the strain just carry tlh. So we can found that the presence of virulence gene could affect the expression of tlh.4. A quantification method of viable Vibrio parahaemolyticus based on RNA technique wasfirst established. This method eliminated enrichment and training process of the conventionalmethod that can be completed within5hours of the experiment. This approach improves the lack ofquantitative methods DNA, interfering exclude dead cells, so it can get more accurate result. Thedesigned tlh primer has a high specificity and the standard curves lopes of standard RNA showingthe good linear relationship. And the limit of quantification was58cfu/g. It could be used in thequantification of seafood samples.