Detection of viable enterohemorrhagic Escherichia coli O157 using the combination of immunomagnetic separation with the reverse transcription multiplex TaqMan PCR system in food and stool samples.
Enterohemorrhagic Escherichia coli O157:H7 is an infectious pathogen and outbreaks have been reported all over the world, specifically in Australia, Canada, Japan, the United States, and in various countries in Europe and South Africa. Therefore, it is necessary to develop rapid methods to determine the target pathogens for food sanitation and disease. Three combinations of primers and probes were designed to detect and identify E. coli O157 using the TaqMan detection system which focuses on the specific genes eae, rfb(O157, and stxII. Reverse transcription (RT) multiplex TaqMan PCR was carried out to accurately detect viable target cells correctly. Furthermore, the acidic pretreatment and immunomagnetic separation (IMS) of food and stool samples also improved the specificity and accuracy of the RT multiplex TaqMan PCR. The developed multiplex TaqMan PCR was effective in differentiating E. coli O157, enterovirulent E. coli, and non-E. coli pathogens from 100 strains which were isolated from clinical patients and the environment. Viable and nonviable cells were also distinguished by this assay. The pretreatment protocol, which included IMS to concentrate and purify the E. coli O157, was developed and the sensitivity of the assay was improved to 10(0) CFU/ml in pure culture, food, and stool samples. The TaqMan PCR assay is a rapid test for the detection of E. coli O157 in food and stool matrices. It shortens the process time and increases the specificity of the pathogens detected. This is critical for improving the safety and sanitation of our food supply.
Tsai
Tsung-Yu
TY
Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan 106, Republic of China.
Lee
Wan-Ju
WJ
Huang
Yu-Ju
YJ
Chen
Kuang-Lo
KL
Pan
Tzu-Ming
TM
D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002470:Cell Survival; D015169:Colony Count, Microbial / Q000379:methods; D003257:Consumer Product Safety; D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry*; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000382:microbiology; D005506:Food Contamination / Q000032:analysis* / Q000517:prevention & control; D005516:Food Microbiology; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000737:chemistry / Q000235:genetics; D022641:Shiga Toxin 2 / Q000737:chemistry / Q000235:genetics; D013045:Species Specificity; D013997:Time Factors
D018829:Adhesins, Bacterial; D015342:DNA Probes; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; C073819:eaeA protein, E coli