Real-time PCR for detection of fliC gene of E. coli O157:H7 in beef and chicken meat

The sybr green i real-time pcr assay was used to quantify e. coli o157:h7 in various meat samples. primers were designed to amplify and quantify the structural flagella (flic) gene of e. coli o157:h7 in a single reaction. the primer specificity was confirmed with dna from an atcc culture of e. coli o157:h7 edl933 as positive control, autoclaved e. coli o157:h7 edl933 as negative control (nc) and nuclease free water as non template control (ntc). a direct correlation was determined between the fluorescence threshold (ct) and the starting quantity of e. coli o157:h7 dna. a detection limit of 4.71 x 10^–2 ng/ μl of e. coli o157:h7 dna equivalent to approximately 1.4 x 10^–2 cfu of e. coli o157:h7 ml^–1 based on plate counts was determined. quantification of e. coli o157:h7 in australian and malaysian beef, chicken meat, burger and minced beef from the markets was possible when dna quantity was as low as 1.0 x 10^–2 ng/μl. these results indicated that the developed pcr assay was suitable for quantitative determination of e. coli o157:h7 in meat samples.