Authors: Wang, Luxin¹; Li, Yong²; Mustapha, Azlin¹

Source: Journal of Food Protection®, Volume 70, Number 6, June 2007 , pp. 1366-1372(7)

Publisher: International Association for Food Protection

Abstract:

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 10² to 10⁹ CFU/ml for E. coli O157:H7, 10³ to 10⁹ CFU/ml for Salmonella, and 10¹ to 10⁸ CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 10⁵ CFU/g for E. coli O157:H7, 10³ CFU/g for Salmonella, and 10⁴ CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 10³ CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli O157:H7, Salmonella, and Shigella in food.

Document Type: Research article

Affiliations: 1: Food Science Program, Division of Food Systems and Bioengineering, 256 WCS Wing, Eckles Hall, University of Missouri, Columbia, Missouri 65211, USA 2: Department of Human Nutrition, Food, and Animal Sciences, University of Hawaii at Manoa, 1955 East-West Road, Agricultural Science 216, Honolulu, Hawaii 96822, USA

Publication date: 2007-06-01

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