Discriminating EHEC & EPEC Subtypes
Infections with E. coli, 026:H11 can cause severe diarrhea and hemolytic-uremic syndrome. Infection severity has been shown to be dependent on pathogen subtype. While EPEC subtypes produce diarrhea in children, the even more dangerous EHEC subtypes can produce bloody inflammation of the bowel, thrombocytopenia, and kidney dysfunction.
EHEC pathogens produced 56 fatalities in an outbreak in Germany in the summer of 2011. Using traditional microbiological and biochemical tests, it is not possible to differentiate EHEC and EPEC subtypes. Genotyping the pathogenic E. coli genes wzxO26, flic, eae-beta, stx, espK, and arcA has been established as the most reliable way to identify most subtypes.
While wzxO26, flic, eae-beta, stx, and espK can be identified using high-throughput qPCR, it was not previously possible to use a routine qPCR method to differentiate EHEC and EPEC subtypes based on the single base variation (SNP) in the E. coli arcA gene. Researchers at the Agence Nationale de Sécurité Sanitaire de l’Alimentation de l’Environnement et du Travail (ANSES), headed by Sabine Delannoy, have recently shown that arcA genotyping using the LightCycler 1536 Real Time PCR System is fast, accurate, and reproducible.
Using this system, 148 E. coli subtypes were tested in duplicate. Each haplotype used had previously been sequenced. At the same time, 33 O26:H11 subtypes of unknown genotype were also characterized. For real-time PCR, the Bravo Liquid Dispensing platform (Agilent) was used to dispense 1 µL volumes per well. The PCR test used primers specifically designed to allow target detection by a MGB (minor groove binder)-conjugated probe, or alternatively, by hydrolysis probes based on locked nucleic acid (LNA) technology.
Altogether, 769 samples were tested in duplicate, with no PCR failures. Detection sensitivity was very high. The 0.1 to 1.0 ng DNA templates used generated crossing point (Cp) values of 19.2 to 27.4 using MGB probes, and 18.9 to 26.3 using LNA probes. Delannoy et al. reported the high accuracy obtained in their results. A 100% correspondence was found for the genotyping results of the 148 samples when comparing results produced by the LightCycler 1536 System and DNA sequencing. Requiring just 90 min, the test was shown to be suitable for rapid high-throughput screening.