Determination Of Pathogenic To-Xins By Flight-Of-Time Mass Spec-Trometry And Analysis Of Toxin Genes By Real-Time PCR

Vibrio parahaemolyticus,staphylococcus aureus,bacillus cereus and enterotoxigenic escherichia coli were prevalent pathogens in environment,and the major virulence factors were thermostable direct hemolysin(TDH),stapylococcal enterotoxin A(SEA),cereulide and heat-stable enterotoxin a(STa)respectively.These pathogens above were harmful to human health,and they would cause immune system diseases through food poisoning.Therefore,there should be necessary to have a fast,accurate and reliable methods to qualitative and quantitative analyzing these pathogens.Methods of molecular biology and analytical chemistry were used to analyzing vibrio parahaemolyticus,staphylococcus aureus,bacillus cereus and enterotoxigenic escherichia coli.Firstly,the technology of real-time fluorescence polymerase chain reaction(PCR)was used to determine genes of tdh,sea,ces and sta for establishing a high throughput,fast,accurate and reliable method of quadruplex real-time fluorescence PCR;secondly,technoogy of high performance liquid chromatography coupled time-of-flight mass spectrometry(HPLC-ESI-tof)was used to analyzing TDH,SEA,cereulide and STa for establishing a fast,simple,accurate,sensitive and reliable method of qualitative and analysis.The specific primer and probe of this four target genes were determined respectively through screening.The four specific primers and probes were added to PCR reaction system for simultaneously detecting four target genes,the result showed that each of the four target genes amplified specially under specific primer and probe.In addition,the four toxins were analyzed by HPLC-ESI-tof,their characteristic molecular ions were found out,and what’s more the ionization and sensitivity were promoted through study of mobile phase and ratio of aqueous phase to organic phase.The method quadruplex real-time PCR for determination of tdh,sea,ces and sta was established in this paper,which had advantage of high throughput determination that it could help saving time for the whole experiment of pathogens detection;and the methods of HPLC-ESI-tof for determination of TDH,SEA,cereulide and STa were established,which had advantages of simple,fast,accurate and high sensitive.Afterwards this paper also provided evidence for determination of pathogens which carry this four toxin genes in real samples.