Analysis Of Virulence Genes Expression Variation In Vibrio Parahaemolyticus

Vibrio parahaemolyticus (Vp) belonging to Vibrionaceae Vibrio, is a Gram-negativehalophilic bacteria, widely distributed in coastal areas, salt lakes, fish, shellfish and otherseafoods. It is an important pathogen causing food poisoning in coastal region.Consumption of seafoods that contaminated with Vibrio parahaemolyticus can causepatients with diarrhea, intestinal cramps, nausea, vomiting, fever, gastroenteritis typicalreaction, severe cases can cause sepsis and even death. Currently, food poisoning causedby Vibrio parahaemolyticus showing a rising trend, it is becoming a primary food-bornepathogens.Present studies suggests that the most important causative factor of Vibrioparahaemolyticus is the toxin produced by a variety of hemolysis, there are thermostabledirect hemolyticus(TDH), tdh-related hemolysin(TRH), themolabile hemolysin(TLH),respectively encoded by tdh, trh and tlh genes, tlh(thermolabile hemolysin) and tdh(heatdirect hemolysin) are major virulence genes of Vibrio parahaemolyticus.Commonly used detection methods of Vibrio parahaemolyticus including traditionalculture methods, immunological methods, PCR and other molecular biology methods. Butthese methods have their own shortcoming in specificity, sensitivity, detection period,operating and cost of testings. Real-time fluorescence quantitative PCR technology islaunched by Applied Biosystems in 1996. It can quantification nucleic acid by detectingchanges of fluorescence signal. It is a technology that Fluorophoreis added in the PCRreaction, the use of accumulation of fluorescent signal throughout the PCR process inreal-time monitoring. It has a more specific, automation, effective solution to PCRcontamination compared with conventional PCR. It has been widely used.The real-time fluorescence quantitative PCR technique used to analyze the virulencegene expression changes of tlh and tdh. Established a detection method for virulenceexpression of tlh and tdh. Indirectly compared the virulence of different strains by comparing the virulence of genes in different conditions. Then study the pathogenicity ofVibrio parahaemolyticus on different environment.In this paper, Three Vp strains under different conditions as material culture. TotalRNA were extracted from them, 16SrRNA as an internal standard gene, detection of geneexpression of tlh and tdh under different conditions using fluorescence quantitative PCR.The results showed that:1. Four RNA extraction methods were compared in the study. Total RNA extractedfrom PureLinkTM RNA Mini Kit has the best quality, with minimal impact on the PCRreaction, Trizol method Followed by it, although total RNA concentration less than the Kit,but the purity comparable with PureLink kit, and PCR results are better. In comparison offour methods: The advantages of PureLinkTM RNA Mini Kit and Uniq-10 column Trizoltotal RNA extraction kit are simple and rapid, but more expensive. Trizol method andSDS method as the traditional classical RNA purification methods, advantages of the twomethods are: samples are cracked more fully, protein digested more completely;disadvantages are they have more extraction steps, reaction time is longer. But thereagents are cheaper. Therefore, these two methods are more suitable for ordinarylaboratory to extract total RNA.2. We established a technology that detection of virulence gene expression in Vibrioparahaemolyticus. All bacterial have16S gene fragment, it is conservative in differentstrain of Vibrio parahaemolyticus, expression of the gene is stable. In this study,16SrRNA as an internal gene, it used to compare the expression differences of tlh and tdh.By introducing an internal standard, we can further determine the amount of RNA andcDNA consistency. Reverse transcription has one-step and two-step reaction, the studycompared the two methods, and ultimately determine the use of two-step reaction. It isdepart reverse transcription reaction and PCR in two steps, avoiding post-test failuresbecause of the poor quality of total RNA, play a good role on controling the wholeexperimental process.3. Using SPSS statistical software to analysis the findings, conclusions followed: (1)factors that affect the expression of tlh: strain> salinity> temperature; (2) factors thataffect the expression of tdh: strain> temperature> salinity. Integrated all the factors, thefollowing conclusions can be drawn: impact of salinity on tlh greater than on tdh; impactof temperature on tdh greater than on tlh; impact of strain on tlh greater than on tdh.4. Comparison of the experimental results: strains, just the internal transcriptionfactors on gene expression most affected. Environmental factors followed by it, includingtemperature, salinity, pH and so on. Different environmental factors affect on gene expression level inconsistency, we can indirectly compare the virulence of the bacteriathough compare the expression of virulence genes. Then change the strain's virulence bychanging the environmental conditions, so as to provide the basis for study thepathogenicity of Vibrio parahaemolyticus, as well as the relationship between geneexpression and environmental adaptability.