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Pathogenicity Analysis And Development Of The PCR Method With Internal Amplification For The Detection Of Vibrio Parahaemolyticus

Posted on:2012-01-08Degree:MasterType:Thesis
Country:ChinaCandidate:M DaiFull Text:PDF
GTID:2211330362959687Subject:Food Science
Abstract/Summary:PDF Full Text Request
Vibrio parahaemolyticus is one of the most important food-borne pathogens that can cause gastroenteritis and traveler's diarrhea in humans after they consume contaminated raw or partially cooked fish or shellfish, particularly oysters. Fish and shellfish are the main vehicles for the transmission of this pathogen. Outbreaks of Vibrio parahaemolyticus gastroenteritis in China, Japan, Southeast Asia and the United States have significantly increased during the past 10 years.Zhoushan and Ningbo in Zhejiang proince are important production bases of seafood in East China, while Pudong and Jinshan in Shanghai have great consumption of seafood. The clinical V. parahaemolyticus isolates (n=158, 2006~2009), which came from Pudong, Jinshan, zhoushan and Ningbo were serotyped,and their virulence-related genes were analyzed in order to predict the distribution and epidemics of this pathogen. We also established a PCR detection system with an IAC (internal amplification control) to test this pathogen in oyster tissues.At first in this work, clinical V. parahaemolyticus isolates were serotyped. The results showed that those isolates were divided into 22 serotypes, and V. parahaemolyticus O3:K6 isolates were the epidemic strains (107/158). Other serotypes of these isolates were O1:K6, O3:K68, O1:K25, O1:K56, O2:K3, and so on.Secondly, virulence-related genes including tdh (encoding thermostable direct hemolysin [TDH]) and trh (encoding TDH -related haemolysin) were analyzed. The results demonstrated that six out of 158 isolates were (tdh+/trh+) strains, 147 isolates were (tdh+/trh—) strains, and 5 isolates were (tdh—/trh—) strains.Thirdly, the PCR detection system with IAC for V. parahaemolyticus was established and evaluated:1) On-line BLAST was used to select a specific target DNA fragment that could specifically detect V. parahaemolyticus. An IAC was constructed using a DNA sequence which was heterologous to target gene. This sequence had been designed and linked on the plasmid.2) This system was tested by V. parahaemolyticus ATCC17802 and V. parahaemolyticus ATCC33846, 158 clinical V. parahaemolyticus isolates, 59 non-V. parahaemolyticus. The results showed that all V. parahaemolyticus could amplify 451 bp fragments.3) Sensitivity of this system was 0.504 fg/μL for the pure genomic DNA and 8.5×10~2 CFU/mL for pure culture, when the IAC was 5.75×10~5 copies/PCR.At last, we did the pre-treatment of oyster sample and detection of this pathogen in oysters.1) The centrifuge (300×g, 1 min) deposited big particles in oyster tissue homogenate, and then we collected bacterial cells from the supernatant.2) The comparison of four methods for DNA recovery indicated that AllMag○R Blood Genomic DNA Kit was more convenient and efficient.3) After four-hour enrichment, we could detect this pathogen in oyster samples. The results were checked by legitimate method.In conclusion, the IAC-PCR detection system was more rapid, sensitive, which could be used in rapid detection of Vibrio parahaemolyticus in food samples.
Keywords/Search Tags:V. parahaemolyticus isolates, serotypes, virulence genes, Internal Amplification Control, PCR
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