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

• Main Authors: Mohd Saad, Suria, Abdul Kadir, Adlin Azlina, Ishak, Zamri, Abdul Talib, Mohd Afendy, Lau, Han Yih, Shamsudin, Mariana Nor, Abdul Rahim, Raha
• Format: Article
• Language: English
• Published: Malaysian Agricultural Research and Development Institute (MARDI) 2012
• Online Access: http://psasir.upm.edu.my/id/eprint/49301/1/Real-time%20PCR%20for%20detection%20of%20fliC%20gene%20of%20E.%20coli%20O157H7%20in%20beef%20and%20chicken%20meat.pdf; http://psasir.upm.edu.my/id/eprint/49301/; http://ejtafs.mardi.gov.my/index.php/publication/issues/archive/64-2012/volume-40-no1/249-400109

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.