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Real-time PCR detection of enterovirulent Escherichia coli

Abstract number: P1979

Andersson M., Vondracek M.

Enterovirulent Escherichia coli are a common cause of morbidity throughout the word. The major categories of pathogenic E. coli include enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and enteroinvasive E. coli (EIEC). Targeting specific genetic elements i.e. vt1 and vt2 (EHEC), eaeA (EHEC/EPEC), bfpA (EPEC), estA and eltB (ETEC) and ial (EIEC/Shigella) we have developed a TaqMan-based real time PCR system to detect these pathogens from primary streaks of stool cultures. Using a common PCR protocol for all the selected virulence genes we were able to detect between 1 and 10 genomic equivalents for all selected targets (except the variant vt2d which was detected at 100 genomic equivalents). The specificity of the reactions was 100% when tested against 32 different bacterial strains commonly present in clinical samples. To validate the protocol both control strains, n = 212 (32 EHEC, 36 EPEC, 97 ETEC and 47 EIEC/Shigella) and primary streaks of stool cultures, n = 139 (62 EHEC, 17 EPEC, 22 ETEC and 38 EIEC/Shigella) were tested and compared to a well established diagnostic gel based PCR protocol. In total, eleven discrepancies were observed; vt1/vt2 (n = 5), estA/eltB (n = 5) and bfpA (n = 1) which all could be explained by the higher sensitivity of the real time protocol. Furthermore, after using the system in routine diagnostics for up to one year the variation of the reactions were around 2 percent (CV = 1.3–2.1%). We were also able to correlate the frequency of isolated strains to the amount of target DNA present in the initial samples. On this basis we have developed a cut off value which likely corresponds to the lowest DNA content representing presence of viable pathogens. This raise the question of how to deal with weakly positive samples from a system based on nucleic acid detection.

In conclusion, we have developed a rapid and robust TaqMan based assay for routine diagnostics of enterovirulent Escherichia coli. Moreover, the method opens up for studies on clinical relevance in correlation to low DNA content in clinical samples.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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