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United States Department of Agriculture

Agricultural Research Service

Research Project: Pathogen Reduction and Processing Parameters in Poultry Processing Systems

Location: Poultry Microbiological Safety Research

Title: Quantification of zoonotic bacterial pathogens within commercial poultry processing water samples using droplet digital PC.

Authors
item Rothrock, Michael
item Hiett, Kelli
item Kiepper, Brian -
item Ingram, Kimberly
item Hinton, Jr, Arthur

Submitted to: Advances in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 2, 2013
Publication Date: September 1, 2013
Citation: Rothrock Jr, M.J., Hiett, K.L., Kiepper, B., Ingram, K.D., Hinton Jr, A. 2013. Quantification of zoonotic bacterial pathogens within commercial poultry processing water samples using droplet digital PCR. Advances in Microbiology. 3:403-411.

Interpretive Summary: Raw poultry and poultry products are a significant source of zoonotic bacterial pathogen transmission; thus the sensitive detection of major zoonotic bacterial poultry pathogens (Salmonella, Campylobacter jejuni, Escherichia coli, and Listeria monocytogenes) is a vital food safety issue. While traditional culture and molecular-based quantitative PCR have been considered the “gold standards” for bacterial pathogen detection within the poultry production chain, both methodologies have their caveats that can limit their efficacy. Recently, third generation PCR technology, known as droplet digital PCR has been developed to more accurately and sensitively detect genetic targets by allowing for ~20,000 individual PCR nanoliter-sized reactions within a single PCR reaction. The goal of the present study was to determine the efficacy of using this new detection technology within the poultry processing environment, and to compare it to the cultural and molecular “gold standard” methods. When analyzing processing water samples from the final scalder tank and chiller tanks at three stages (Start, Mid, and End) of processing over three consecutive days, ddPCR detected genes specific to each of the targeted bacterial pathogens from more pathogen:sampling time combinations than either the qPCR or traditional culturing methods. In fact, ddPCR far outperformed the traditional culture methods commonly used in most poultry processing food safety-related studies, considering only Salmonella was recovered culturally from the Mid and End sampling times from the scalder tank. Additional analyses comparing the two molecular-based methods demonstrated that the use of unfiltered processing water provided significantly (P < 0.05) greater detection than did analysis of larger volumes of filtered water. Considering the concentrations of the targeted bacterial pathogens using ddPCR were consistent with what has been previously found in commercial poultry processing operations, further validation of ddPCR for zoonotic pathogen detection and quantification is highly recommended.

Technical Abstract: Raw poultry and poultry products are a significant source of zoonotic bacterial pathogen transmission; thus the sensitive detection of major zoonotic bacterial poultry pathogens (Salmonella, Campylobacter jejuni, Escherichia coli, and Listeria monocytogenes) is a vital food safety issue. While traditional culture and molecular-based quantitative PCR have been considered the “gold standards” for bacterial pathogen detection within the poultry production chain, both methodologies have their caveats that can limit their efficacy. Recently, third generation PCR technology, known as droplet digital PCR has been developed to more accurately and sensitively detect genetic targets by allowing for ~20,000 individual PCR nanoliter-sized reactions within a single PCR reaction. The goal of the present study was to determine the efficacy of using this new detection technology within the poultry processing environment, and to compare it to the cultural and molecular “gold standard” methods. When analyzing processing water samples from the final scalder tank and chiller tanks at three stages (Start, Mid, and End) of processing over three consecutive days, ddPCR detected genes specific to each of the targeted bacterial pathogens from more pathogen:sampling time combinations than either the qPCR or traditional culturing methods. In fact, ddPCR far outperformed the traditional culture methods commonly used in most poultry processing food safety-related studies, considering only Salmonella was recovered culturally from the Mid and End sampling times from the scalder tank. Additional analyses comparing the two molecular-based methods demonstrated that the use of unfiltered processing water provided significantly (P < 0.05) greater detection than did analysis of larger volumes of filtered water. Considering the concentrations of the targeted bacterial pathogens using ddPCR were consistent with what has been previously found in commercial poultry processing operations, further validation of ddPCR for zoonotic pathogen detection and quantification is highly recommended.

Last Modified: 10/1/2014