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Applied and Environmental Microbiology, June 2009, p. 3714-3720, Vol. 75, No. 11
0099-2240/09/$08.00+0     doi:10.1128/AEM.02686-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Detection of Live Salmonella sp. Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA{triangledown} ,{dagger}

Narjol González-Escalona,* Thomas S. Hammack, Mindi Russell, Andrew P. Jacobson, Antonio J. De Jesús, Eric W. Brown, and Keith A. Lampel

Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland

Received 24 November 2008/ Accepted 3 April 2009

Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponential-, late-exponential-, and stationary-phase cells. This assay has a detection limit of 40 copies of invA mRNA per reaction. The levels of invA mRNA in mid-exponential-, late-exponential-, and stationary-phase S. enterica cells was approximately 1 copy per 3 CFU, 1 copy per CFU, and 4 copies per 103 CFU, respectively. Spinach, tomatoes, jalapeno peppers, and serrano peppers were artificially contaminated with four different Salmonella serovars at levels of 105 and less than 10 CFU. These foods were analyzed with qRT-PCR and with the FDA's Bacteriological Analytical Manual Salmonella culture method (W. A. Andrews and T. S. Hammack, in G. J. Jackson et al., ed., Bacteriological analytical manual online, http://www.cfsan.fda.gov/~ebam/bam-5.html, 2007). Comparable results were obtained by both methods. Only live Salmonella cells could be detected by this qRT-PCR assay, thus avoiding the dangers of false-positive results from nonviable cells. False negatives (inhibition of the PCR) were also ruled out through the use of an RNA internal control. This assay allows for the fast and accurate detection of viable Salmonella spp. in spinach, tomatoes, and in both jalapeno and serrano peppers.

* Corresponding author. Mailing address: Center for Food Safety and Applied Nutrition, FDA, 5100 Paint Branch Parkway HFS-712, College Park, MD 20740. Phone: (301) 436-1937. Fax: (301) 436-2644. E-mail: narjol.gonzalez-escalona{at}fda.hhs.gov

{triangledown} Published ahead of print on 17 April 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, June 2009, p. 3714-3720, Vol. 75, No. 11
0099-2240/09/$08.00+0     doi:10.1128/AEM.02686-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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