| Diarrheagenic Escherichia coli(DEC)is an important foodborne pathogen that can easily spread and cause infection in humans.DEC infection may lead to a series of diseases such as abdominal pain,diarrhea,hemorrhagic colitis,and hemolytic uremic syndrome.As a significant public health issue,its rapid and accurate detection has become an important concern for food safety issues and healthcare-associated infections.We have developed a DEC rapid detection kit based on multiplex real-time quantitative PCR assay(qPCR),to simultaneously recognize five major DEC pathotypes in a single tube,enterotoxigenic E.coli,enterohemorrhagic E.coli,enteropathogenic E.coli,enteroaggregative E.coli,and enteroinvasive E.coli.In this assay,13 DEC virulence genes,stp,sth,It,stx1,stx2,eae,escV,bfpB,aggR,astA,pic,invE,and ipaH,can be simultaneously detected along with a spiked-in internal amplification control(IAC)to avoid false negative results.The first chapter briefly described the development process of DEC rapid detection kit,including the selection of target genes,the design of primer probes,the optimization of single and multiple qPCR and the experimental results.The second chapter first explained the discovery and confirmation of cross contamination among commercial plasmids,and then decribed the performance evaluation of DEC rapid detection kit,including detection sensitivity,specificity and resistance to interference.The results showed that the detection sensitivity of the kit was high with a detection limit of about 10 copies per reaction,and the PCR efficiency ranged from 96%to 110%.The assay was also demonstrated to be highly quantitative with excellent linearity(R2≥0.99)and excellent specificity.Lastly,when two different targets were present in the same reaction at a concentration difference of 10,000-fold,the Ct values of the low-concentration targets were only delayed for no more than 2.3 in comparison to those in the absence of competing targets,equivalent to a detection error within 5 fold,demonstrating a robust resistence of individual reactions against the concurrent competing amplification reactions.The third chapter depicted the application of DEC rapid detection kit,including successful discrimination between live and dead bacteria and the detection of DEC from randomly picked food samples.In the simulation of live bacteria detection,the kit can successfully recognize only 0.01%of live bacteria from dead ones within 4 hours.In the real life application,219 randomly picked food samples were tested,and 20 of them were scored positive by DEC with a contamination rate of as high as 9.1%,further demonstrating the importance of foodborne pathogen detection and the necessity of high temperature sterilization of raw foods.In summary,we have successfully developed a DEC rapid detection kit using multiplex real-time quantitative PCR.It can detect at the same time 13 virulence genes characteristic of five DEC pathotypes,and has the advantages of high sensitivity,excellent specificity,and resistance to interference.It has been successfully applied to DEC contamination detection of food samples.It also has the potential to be used for clinical samples in the future as a diagnostic tool.Its popularization and application may become a powerful tool to prevent and fight DEC-associated diseases and to improve public health. |
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