Rapid quantification of escherichia coli as an indicator of food contamination using fluorescence in situ hybridization with filter cultivation(FISHFC)

Abstract

Conventional plate counting for Escherichia coli is time consuming, and more rapid and specific quantification methods are desired. The fluorescence in situ hybridization with filter cultivation (FISHFC) method has already been developed to specifically detect viable microorganisms such as Listeria monocytogenes, Clostridium perfringens and Staphylococcus aureus. The purpose of this study was to develop and estimate the accuracy of the FISHFC method for E. coli detection in food samples. An Alexa FluorR 647-labeled oligonucleotide probe, ECO636, targeting a species-specific sequence of E. coli 16SrRNA was designed. ECO636-conferred fluorescence was observed for E. coli and Shigella spp. micro-colonies, but not for any other organisms. This suggests that the ECO636 probe is highly specific for E. coli and Shigella spp. This assay using the FISHFC method can be completed within 9 hours, including a 7-hour cultivation period, and is much faster compared to standard plating methods, which require more than 4 days, for the confirmation of E. coli. Viable E. coli counts in food samples determined by the FISHFC method were not significantly different to those obtained by the conventional plate counting method (p>0.05). These results suggest that the proposed FISHFC method is more rapid than and equally as reliable as the standard methods used for estimating viable E. coli numbers as indicators of food contamination in food.