12 January - 20 June 2016

Use of Flow Cytometry for detection of E.coli

13 January 2016

In an article published in the International Journal of Food Microbiology, a team from the US Food and Drug Administration published the results of a validation exercise for the detection of E.coli. O157:H7 in raw spinach by Flow Cytometry (FC). Such enterohemorrhagic strains of E.coli are of concern as they are particularly virulent with as few 1 – 10 colony forming units (CFU’S) having the potential of causing diarrheal illness, with a high mortality rate seen in elderly patients with up to 50% succumbing to the illness.

The current method for E.coli analysis in the Bacteriological Analytical Manual (BAM) specifies the use of PCR for the detection of E.coli. For a new method to be used it must be shown to be equivalent or better to the compendial method. A level 2 validation was performed in which a double blind study was carried out across both techniques. The flow cytometer used in this study can quickly detect and enumerate a few cells of E. coli O157, differentiating them from other serotypes. For the validation 40 samples of raw spinach were obtained, 20 of which were spiked with low levels of E. coli O157:H7, the remaining 20 samples were left clean. Each technique then analysed 10 unspiked samples and 10 spiked samples. After the analysis it was clear that both techniques were comparable in terms of detection, with the FC technique detecting 60% of the positive samples, compared to 50% for the PCR methodology. The high number of false negatives seen may be due to the low level of spiking used mean that no viable E.coli cells were inoculated.

The key benefit for use of the FC technique is the massive gain in time, since the complete analysis for the validation using the FC technique was 9h whereas the PCR technique took 51h, which has a clear advantage in terms of shelf-life of the product after testing as well as logistically in terms of production and distribution.

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