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Applied and Environmental Microbiology, January 2007, p. 92-100, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.01772-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rapid Separation and Concentration of Food-Borne Pathogens in Food Samples Prior to Quantification by Viable-Cell Counting and Real-Time PCR{triangledown}

Hiroshi Fukushima,* Kazunori Katsube, Yukiko Hata, Ryoko Kishi, and Satomi Fujiwara

Shimane Prefectural Institute of Public Health and Environmental Science, 582 Nishihamasada, Matsue, Shimane 690-0122, Japan

Received 26 July 2006/ Accepted 11 October 2006

Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low- and high-speed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Vibrio cholerae O139, Vibrio parahaemolyticus O3K6, Vibrio vulnificus, Providencia alcalifaciens, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 101 to 103 CFU/g using the RTi-qPCR assay, and amounts as small as 100 to 101 CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 101 to 102 CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak.

* Corresponding author. Mailing address: Shimane Prefectural Institute of Public Health and Environmental Science, 582 Nishihamasada, Matsue, Shimane 690-0122, Japan. Phone: 0852-36-8181. Fax: 0852-36-8171. E-mail: fukushima-hiroshi{at}pref.shimane.lg.jp.

{triangledown} Published ahead of print on 20 October 2006.

Applied and Environmental Microbiology, January 2007, p. 92-100, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.01772-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Monnet, C., Ulve, V., Sarthou, A.-S., Irlinger, F. (2008). Extraction of RNA from Cheese without Prior Separation of Microbial Cells. Appl. Environ. Microbiol. 74: 5724-5730 [Abstract] [Full Text]  


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