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United States Department of Agriculture

Agricultural Research Service

Research Project: INTEGRATED BIOSENSOR-BASED PROCESSES FOR MULTIPATHOGENIC ANALYTE DETECTION Title: A multiplex real-time PCR for simultaneous detection of Salmonella spp., E. coli O157 and L. monocytogenes in meat products

Authors
item Suo, Biao -
item He, Yiping
item Tu, Shu-I
item Shi, Xianming -

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 10, 2010
Publication Date: June 1, 2010
Citation: Suo, B., He, Y., Tu, S., Shi, X. 2010. A multiplex real-time PCR for simultaneous detection of Salmonella spp., E. coli O157 and L. monocytogenes in meat products. Foodborne Pathogens and Disease. 7(6):619-628.

Interpretive Summary: Foodborne illnesses are often caused by consumption of food contaminated by microbial pathogens. Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes are the most prominent microbial pathogens implicated in foodborne illnesses. To effectively monitor and control microbial contamination in food, we have developed a rapid and sensitive method for simultaneous detection of these three pathogens in meat samples. The assay combined a multi-pathogen enrichment strategy with multi-target identification to achieve multiple pathogen detection in a single assay. To control false-negative results, a novel internal control was designed and incorporated into the assay. The performance of the assay was thoroughly evaluated in both artificially and naturally contaminated meat samples. Overall, the assay developed in this study has a high degree of specificity, sensitivity and reliability for effective detection of the three pathogens in food.

Technical Abstract: To achieve multipathogen detection in a single platform assay, a multiplex real-time PCR assay was developed for simultaneous detection of Salmonella spp., E. coli O157 and L. monocytogenes in food. Pathogen specific DNA sequences in the invA, rfbE and hlyA genes were employed to design primers and TaqMan probes for identifying Salmonella spp., E. coli O157 and L. monocytogenes, respectively. A novel Internal Amplification Control (IAC) utilizing an exclusive DNA sequence from human adenovirus was incorporated into the multiplex PCR assay to indicate false-negative results. Concurrent amplifications of multiple targets and IAC were thoroughly evaluated and optimized to minimize PCR competitions. Combined with a multipathogen enrichment strategy, the multiplex real-time PCR assay was able to simultaneously detect all of the three pathogens in artificially contaminated ground beef at detection sensitivity of 18 CFU/10 g ground beef or even lower. Applying the assay to 26 retail meat samples revealed that 12 samples (46.2%) were positive for one of the pathogens and 3 samples were positive for two of the pathogens after 20-hr enrichment in a multipathogen selective enrichment broth (SEL). All of the pathogen-negative meat samples were indicated to be true negatives because the IAC amplification occurred in the same reaction tubes. These results were confirmed by traditional culture methods in analyzing each individual species. Taken together, the multiplex real-time PCR assay combined with multi-pathogen enrichment is a rapid and reliable method for effectively screening single or multiple pathogen occurrences in various meat products.

Last Modified: 11/27/2014
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