Establishment Of Rapid Detection System By PCR And Quantification Study On Pathogen Of Vibrio Parahaemolyticus In Seafood

Vibrio parahaemolyticus is a Gram-negative halopilic bacterium distributed widely in the estuarine environment, especially in coastal fish, shellfish and seafood products. Pathogenic V. parahaemolyticus strains caused acute gastroenteritis, such as diarrhea, vomiting, abdominal cramps and so on, in humans after consumption of contaminated foods, most often involving unproperly cooked seafoods. In resent years, outbreaks of food poisoning caused by pathogenic V. parahaemolyticus have been reported in various geographic regions, including Japan, China, New Zealand, Indonesia and the USA. More recently, the occurrence of V. parahaemolyticus isolates from environments or seafoods not only in coastal areas but inland has shown an increasing tendency. The current research thinks that vibrio parahaemolyticus’s virulence factors are adhesion factor, aggressie, hemolytic toxins, urease, lipopolysaccharide, exocellular enzymes, secrete systemⅢand perturbation iron system etc, which hemolytic toxin is the most important virulence factors. Hemolytic toxin mainly includes thermostabile direct hemolysin, TDH-related hemolysin and thermolabile hemolysin, which are encoded by relevant gene of tdh, trh and tlh respectively. TDH and TRH have close relationship with the disease-causing ability of vibrio parahaemolyticus. More than 90% strains separated in clinic are TDH positive, however, TDH positive V. parahaemolyticus separated in environment is only 1% to 2%.Ttraditional PCR and real-time PCR technology to make the qualitative and quantitative study of of pathogen viable cells of Vibrio parahaemolyticus in seafood was conducted, and a fast, sensitive new method which could effectively identify and quantitative detection pathogen viable cells was developed. This research including three parts as follows:The first part:Firstly, template DNA extraction was optimized respectively using TZ lysing solution, boil, CTAB/NaCl and TENS, and 16SrDNA fragment of bacteria as internal amplification control(IAC) primers were designed to detect the genes of thermolabile hemolysin (tlh) and thermostable related hemolysin (trh) in vibrio parahaemolyticus, and the assay was optimized. The experiment of specificity and sensitivity showed that this multiplex PCR assay had a good specificity, and for pure culture, the detection limit of tlh and trh was 1.3×102 CFU/ml and 1.3×103 CFU/ml respectively in the presence of IAC; for samples of artificially contaminated oysters, however, the detection sensitivity of tlh and trh was 2.6×103 CFU/ml and 2.6×104 CFU/ml respectively in the presence of IAC without enrichment, and after 6 h enrichment as little as 2.6×102 CFU/ml of tlh and trh could be detected by this multiplex PCR. Results showed that the detection system had a good specificity and sensitivity. The existence of IAC could successfully eliminate false-negative results during using PCR technique to detect pathogenic vibrio parahaemolyticus and could improve the detection speed and accuracy.The second part:A new and efficient method for detection the viable and dead cell of pure cultured of vibrio parahaemolyticus was developed by using a DNA dye of ethidium bromide monoazide (EMA) in combination with the traditional polymerase chain reaction (EMA-PCR). The results showed that, under the light exposure for 20 minutes to photolyse the EMA in cell suspension of 4×108 CFU/ml vibrio parahaemolyticus treated with EMA at the concentration of 1.4μg/ml, the PCR results were negative, but the control without treatment by using EMA, the PCR results were positive. The PCR for viable cell of vibrio parahaemolyticus was not inhibited with the maximum concentration of EMA at 6μg/ml. After EMA treatment, the number of viable vibrio parahaemolyticus cells in varying ratios of viable to dead cells could be selectively quantified by PCR. The minimum level of detection was 10 CFU per PCR reaction. A linear relationship was found between the relative fluorescent intensity of the DNA bands and the log of genomic targets derived from the viable cells in mixtures of viable and dead cells in the range of 10 to 2×105 CFU per PCR reaction.The third part:Firstly the real-time fluorescence quantitative PCR system is optimized, and then a new method for selectively quantitative detection of trh-positive viable cells of Vibrio parahaemolyticus in oysters was developed using ethidium bromide monoazide (EMA) in combination with real-time PCR (RT-PCR, real-time polymerase chain reaction). The results showed that the optimized light exposure time to achieve crosslinking to DNA by the EMA in dead cells and to photolyse the free EMA in solution was 20 min. The EMA concentration of 2.0μg/ml or less did not inhibit the RT-PCR amplification of DNA derived from viable cells of Vibrio parahaemolyticus. The minimum amount of EMA to completely inhibit the RT-PCR amplification of DNA derived from heat-killed cells was 1.4μg/ml. For pure culture, there was a strict inverse correlation between the log of the number of cells and the associated Ct values in the range of 22～2.2×107 CFU, and the detection sensitivity of without EMA(22 CFU) was slightly higher than that of adding EMA(2.2×102 CFU). Artificial contamination of oyster samples, using RT-PCR, EMA RT-PCR and plate counts separately to quantitative detection of Vibrio parahaemolyticus, the analysis comparison indicated that the results of EMA RT-PCR was closer to plate counts and the number derived from RT-PCR was statistically higher than the number obtained from EMA RT-PCR and the plate count. The experiment of freezing and thawing showed that when freezed Oyster samples were thawed at below 55℃the freeze-thaw process had little effect on viable cells of V parahaemolyticus. Artificial contamination of oyster samples and without enrichment, there was a strict inverse correlation between the log of the number of cells and the associated Ct values in the range of 4.7×102～4.7×106 CFU, and the detection limit of the real-time PCR assay was 4.7×102 CFU for artificial contamination of Oyster samples, suggesting that the sensitivity of RT-PCR was 94 CFU/g oyster sample for artificial contamination. After 6 h enrichment, V. parahaemolyticus in pure cultures or Oyster homogenates containing an initial inoculum of 47 CFU, could be detected, and there was no significant difference in Ct values between pure cultures and Oyster homogenates. No linear relationship between the Ct values and log bacterial concentration was observed. The sensitivity of the real-time PCR detection of V. parahaemolyticus in the presence of background flora was examined by combining various dilutions of V. parahaemolyticus with an overnight enrichment culture generated using an uninoculated oyster sample. The total aerobic plate count from the overnight enrichment culture was 8.2×108 CFU/ml. Even in the presence of background flora, real-time PCR was capable of detecting as few as 4.7×102 CFU/ml of V parahaemolyticus. The real-time PCR method was able to detect the V. parahaemolyticus of more than 4.7×102 CFU/ml even in the presence of the background flora. Finally, a total of fourty-five oyster samples were tested for the trh positive strains of viable V. parahaemolyticus by real-time PCR after EMA treatment. Test results showed that only one oyster samples was trh positive of V. parahaemolyticus without enrichment, approximately at a mean bacteria concentration 114 CFU/g.This study provides both rapid and accurate new method for selectively qualitative and quantitative detection of trh-positive viable cells of Vibrio parahaemolyticus in seafoods, and also provides new ideals for detecting viable cells of other samples.