A Multiplex PCR Assay for Simultaneous Detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in Korean Ready-to-Eat Food

Abstract

A multiplex polymerase chain reaction (PCR) assay was developed for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in various Korean ready-to-eat foods. The six specific primer pairs for multiplex PCR were selected based on the O157 antigen (rfbE) gene of E. coli O157:H7, the DNA gyrase subunit B (gyrB) gene of B. cereus, the toxin regulatory protein (toxR) gene of V. parahaemolyticus, the invasion protein A (invA) gene of Salmonella spp., the hemolysin (hly) gene of L. monocytogenes, and the thermonuclease (nuc) gene of S. aureus. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity assays for multiplex primer pairs were investigated by testing different strains. When this multiplex PCR assay was applied to evaluate the validity of detecting six foodborne pathogens in artificially inoculated several ready-to-eat food samples, the assay was able to specifically simultaneously detect as few as 1 colony-forming unit/mL of each pathogen after enrichment for 12 h. Their presence in naturally contaminated samples also indicates that the developed multiplex PCR assay is an effective and informative supplement for practical use.

Microbiological safety of ready‐to‐eat foods in low‐ and middle‐income countries: A comprehensive 10‐year (2009 to 2018) review

“…Such advanced infrastructures will gradually erode the application of conventional techniques (such as, culture‐based microbial isolations and thin‐layer chromatography for toxin detection). Rapid techniques involving immunoassays and nucleic acid‐based methods for foodborne pathogen detection (Lee et al., ) and liquid chromatography–mass spectrometric methods for microbial toxin analysis (Berthiller et al., ) should be adopted to enable accurate and high‐throughput analysis.…”

Listeriosis in animals, its public health significance (food-borne zoonosis) and advances in diagnosis and control: a comprehensive review

“…Molecular tools such as PCR, multiplex PCR and realtime PCR employing virulence-associated genes such as the mpl gene, prfA gene (Rossmanith et al 2006) and ssrA gene (O'grady et al 2008) have been found rapid, specific, reproducible and reliable (Portnoy et al 2002;Gianfranceschi et al 2013;Hage et al 2014;Khan et al 2014). Several multiplex PCR assays have been developed for simultaneous detection of various food-borne pathogens like Salmonella, Escherichia coli, Staphylococcus and also Listeria (Park et al 2006;Kawasaki et al 2009;Lee et al 2014). Multiplex real-time PCR assay based on molecular beacon chemistry was developed recently which could detect eight food-borne pathogens (Salmonella enterica subsp.…”

Development of a multiplex event-specific PCR assay for detection of genetically modified rice

“…Primer design is critical in the multiplex PCR assay for the simultaneous amplification of multi-target DNA sequences to ensure specificity and sensitivity and to avoid crossreactions (Lee et al 2014). As the number of primers increases, the interaction effects also increase and may result in the formation of amplification artifacts, such as primer-dimers, truncated DNA fragments, non-specific bands or an absence of bands (Shrestha et al 2010).…”

“…Although specific primers amplify target DNA sequences, they do so with the differential amplification rates (Kim et al 2009). Taking into account these factors, lengths of multiple sets and melting temperatures (Tm) of primers must be similar Lee et al 2014). To establish a multiplex PCR method for simultaneous detection of three events of GM rice, we designed event-specific primers targeting to three GM rice events, namely, TT51-1, KMD1 and KF6.…”