Development of an Internal Amplification Control Using Multiplex PCR for the Detection of Listeria monocytogenes in Food Products |
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Authors: | Diane Rip Pieter A Gouws |
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Affiliation: | (1) Food Microbiology Research Group, Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville, 7535, South Africa |
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Abstract: | The bacterial pathogen Listeria monocytogenes is responsible for listeriosis, a food-borne disease, which may result in severe illness and possible death. Large outbreaks
of listeriosis have been associated with food products including soft cheeses and ready to eat food products. Polymerase chain
reaction (PCR) is a molecular identification method for food-borne pathogens; however, a drawback of this method is that false-positive
or false-negative results may occur. To validate the accuracy of the PCR as a powerful molecular tool for pathogen detection,
it is important that false-negative results be distinguishable from true-negative PCR results. The aim of this study was to
design and include an internal amplification control (IAC) within the PCR to coamplify with L. monocytogenes in order to identify false-negative results of L. monocytogenes from ostrich meat and camembert cheese samples. The IAC had to be incorporated into the PCR without loss of specificity and
sensitivity on the detection limit of L. monocytogenes and was developed and tested for use in a multiplex PCR detection system. A region of the pUC19 plasmid was selected as the
IAC for this study. The optimal concentration at which pUC19 would coamplify with L. monocytogenes was determined to be 0.001 pg/μL. Following an enrichment procedure, the minimum number of organisms detected in a spiked
food sample by the PCR was 8 CFU/mL L. monocytogenes; the same detection limit was attained when the pUC19 IAC was included in the PCR. An optimal pUC19 IAC concentration increased
the reliability of the PCR for food diagnostic purposes. |
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Keywords: | Internal Amplification Control Listeria monocytogenes PCR Food Products Optimization |
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