Establishment And Application Of Nucleic Acid-Based Detection Methods For Vibrio Parahaemolyticus

Virio parahaemolyticus is primarily involved in causing gastrointestinal illnesses. V. parahaemolyticus is considered to be the causative agent in50%to70%of all cases of diarrhea associated with the consumption of fishery products. Once introduced into food-processing plants, bacteria can persist for a long time. So to dectect V. parahaemolyticus is of great significance. The biochemical identification and enumeration of Vibrio parahaemolyticus as described in the FDA Bacteriological Analytical Manual is expensive and labour-intensive. Futhermore, V. parahaemolyticus can enter a viable but nonculturable (VNC) state. To reduce the time and effort necessary to verify the identity of V. parahaemolyticus, the rapid and sensitive detection of V. parahaemolyticus is necessary. Nucleic acid-based tests have emerged as a useful alternative to traditional testing for the maintenance of a safe seafood supply. In the present study, we have developed a series of nucleic acid based detection methods, including a traditional PCR and a digoxigenin (DIG)-labelled probe method, for the screening of seafood for V. parahaemolyticus. As well, a new technique based on Real-time PCR for quantitative detection V. parahaemolyticus and a multiplex real-time PCR for simultaneous and quantitative detection of V. parahaemolyticus also were established. We then carried out a screening of seafood in Eastern China for the presence of V. parahaemolyticus was carried out base on the mutiplex Real-time PCR and culture method.1Detetection of Vibrio parahaemolyticus in food by traditional PCRThe purpose of this study is to establish a new method for detection V. parahaemolyticus faster and validate its practicability. The new technique involves designing a primer pair targeting tdh gene and using the primer pair for PCR amplification. The method was tried in4V. parahaemolyticus strains and9non-V. parahaemolyticus strains, and also artifically contaminated food samples. The results showed that the technique offered excellent differentiation and lower detection limit (10CFU/g). The full course of assay could be completed in13hours, which was significantly shorter than the time needed by conventional techniques. It is concluded that the technique is practical with advantages of simple operation, higher specificity, less time consuming and lower detection limit. 2A digoxigenin (DIG)-labelled probe for detection of V. parahaemolyticusA digoxigenin (DIG)-labelled probe targeted to the tdh gene fragment was prepared by PCR and evaluated for specificity against5strains of V. parahaemolyticus,2strains of non-vibrio species. Of the7isolates tested, the probe hybridized only with the5strains of V. parahaemolyticus, indicating species specificity. The results suggest that the probe is useful for detection of the V. parahaemolyticus tdh gene.3Application of Real-time PCR for quantitative detection of V. parahaemolyticusIt is important to develop a nucleic acid-based test for quantitative detection of V. parahaemolyticus. A TaqMan PCR assay was presented for quantitative detection of V. parahaemolyticus in pure cultures and oysters. The primers and probe were designed according to the gyrase B gene (gyrB) sequence of V. parahaemolyticus strains on GENEBANK website. Amplification of DNAs from12bacterial strains comprising9genera showed that all of the strains of V. parahaemolyticus tested (n=4) were positive and all other species of strains tested (n=8) were negative.The results of the TaqMan PCR with raw oysters inoculated with V. parahaemolyticus were comparable to those of pure cultures. The sensitivity of the assay was1CFU PCR Mixture-1in pure culture and10CFU PCR mixture-1in inoculated raw oyster, the correlation rate was0.99(r2=0.99). The assay could be completed within1h. The TaqMan probe and primers set developed in this study can be used as a rapid screening tool for the presence of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.4Multiplex Real-Time PCR for quantitative detection of V. parahaemolyticus and Staphylococcus aureusA multiplex Real-time PCR assay was developed for the simultaneous and quantitative detection of V. parahaemolyticus and Vibrio vuLnificus in a single tube. The assay was tested on V. parahaemolyticus (n=10),S. aureus (n=9) and other bacteria species, the result showed that the assay was highly specific for V. parahaemolyticus and S. aureus tested. The sensitivity of the assay was demonstrated to less than10copies of bacteria genome DNA of V. parahaemolyticus and S. aureus, respectively. Quantitative linearity of multiplex Real-time PCR was achieved by amplified ten-fold dilutions of purified genomic DNA of V. parahaemolyticus and S. aureus ranging from107to103CFU mL-1based on plate counts, respectively, the log cell number of bacteria and the amount of production (presented by Ct) showed excellent correlation (r2=1.00). Inhibition studies for the multiplex Real-time PCR assay, performed by spiking the DNA extracts from11negative bacteria with purified DNA from V. parahaemolyticus and S. aureus, these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. These data also indicate that multiplex Real-time PCR can provide sensitive species-specific detection and quantification of V. parahaemolyticus and S. aureus in seafood.5Comparative study on detecton methods for V. parahaemolyticusA comparative study was conducted to determine the efficiency, time consuming and cost of different molecular detction methods for detection for V. parahaemolytiucs from seafood.150samples of seafood, purchased from several local markets in harvest season in Nanjing, were subjected to traditional culture methods, conventional PCR, digoxigenin (DIG)-labelled probe and Real-time PCR assays, respectively. The result was consistent among three molecular methods. The positive ratio for the V. parahaemolyticus detected by three molecular methods was slightly higher than traditional culture methods. However, time consuming and cost is different. The data showed that conventional PCR is more fit for qualitive detection of V. parahaemolyticus from seafood in small number than digoxigenin (DIG)-labelled probe. Digoxigenin (DIG)-labelled probe is suitable for large scale screening of V. parahaemolyticus from seafood. The Real-time PCR can be used as a quantitative tool for the dectiom of V. parahaemolyticus in seafood. The study aslo implicated that the combination of Digoxigenin (DIG)-labelled probe methods and Real-time PCR assay was efficent for large scalely quantitative dection of V. parahaemolyticus.6Study on the distribution of V. parahaemolyticuand in the seafood in Eastern ChinaAccording to the data collected by the National Fodbome Diseases Surveillance Network, the food poisoning caused by V. parahaemolyticus is going up in China recent years. In order to get more information about the V. parahaemolyticus contamination,882samples, including sea shellfish (n=340), shrimp (n=284) and fish (n=258) were collected from several local markets in harvest season in Eastern China within the period from January2005to December2006. The V. parahaemolyticus in seafood samples was determined qualitatively and quantitatively by the digoxigenin (DIG)-labelled probe and Real-time PCR technique. V. parahaemolyticus was isolated from33.1%of all the samples (292/882). High frequency of V. parahaemolyticus was detected in seafoods. Incidence of V. praahaemolyticus in shellfish, shrimp and fish were44.1%,20.4%and29.6%, respectively, with the mean densities of V. parahaemolyticus in positive samples1.0×105/g,7.0×104/g and5.0×104/g, respectively. V. parahaemolyticus was isolated from11.3%of all the water samples (17/150). In addition, the result revealed that the ebb and flow of V. parahaemolyticus related closely to the season. This was consistent to the phenomenon of food poision causing by V. parahaemolyticus. It is concluded that this organism needs to be intensively monitored and controled in raw seafoods.