Comparison between O serotyping method and multiplex real-time PCR to identify diarrheagenic Escherichia coli in Taiwan.

To compare the diarrheagenic Escherichia coli (DEC) identifications obtained between traditional O serotyping and modern virulence gene detection assays, we developed a multiplex real-time PCR assay by detecting six specific virulence genes for enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). Among 261 clinical diarrheal stool samples, a total of 137 suspected DEC (sDEC) isolates were identified by the use of commercially available antisera. The most prevalent serogroups were O1 (12/137; 8.7%), O25 (9/137; 6.5%), and O44 (9/137; 6.5%). The specific virulence genes for the 137 sDEC isolates were analyzed by the multiplex real-time PCR assay. Fifteen (10.9%) of 137 isolates were confirmed to be true DEC strains, indicating that the serotypic markers did not correlate with the specific virulence genes. ETEC (66.7%) was the most prevalent, followed by EIEC (20%) and EPEC (13.3%). No EHEC strains were identified in the specimens. Four novel serotypes were found in the study: two in EPEC strains (O111:H9 and O63:H6) and two in EIEC strains (O63:H9 and O169:H9). In conclusion, the real-time PCR assay considerably reduces the high false-positive rate from the use of serotyping alone, and thus, it is suggested that serogrouping-based methods are inadequate for the identification of DEC isolates, although they are useful for the identification of a limited number of serogroups. In addition, ETEC, EPEC, and EIEC strains were present in 5.7% (15/261) of the diarrheal patients in northern Taiwan in 2006.

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.