Application of multiplex real-time polymerase chain reaction assay for simultaneous quantification of Escherichia coli virulence genes in oysters

Abstract

Strains of diarrheagenic (DEC) are involved in foodborne disease outbreaks worldwide, especially the enterohemorrhagic O157:H7. This study describes two multiplex quantitative real time PCR (qPCR) assays for simultaneous identification and quantification of genes related to virulence of DEC; a triplex reaction for detection and quantification of ,, and genes, and a duplex reaction for detection and quantification of and genes. The technique was applied in raw oyster samples for direct quantification of DEC, thereby evaluating the applicability of this methodology for microbiological quality assessment of food. Using custom designed primers and specific MGB probes, a qPCR assay was performed to quantify ,, and , and a reaction was performed to quantify and genes. The assays showed high sensitivity, with the detection limit varying between 5 and 17 copies of the genes. The coefficient of determination (R) of the standard curves was 0.99. The coefficient of variation was < 1% indicated high intra- and inter-assay reproducibilities. The application of this methodology in oyster samples from tropical environment provided direct quantitative data that determined the presence of the genes (32.1%), (28.6%), (3.6%), and (3.6%). This would prove critical for immediate intervention of control strategies, particularly in oysters that are often ingested as raw food.

Effects of adding a prebiotic product based of beta-glucans, glucomannans, and mannan-oligosaccharides on performance and health of weanling pigs

“…Pathogenic strains are of great importance in pig farming, as they can cause enteritis and PWD. What differentiates the native strain from the pathogenic one is the presence of virulence factors in the latter (Maciel et al, 2018). However, even non-pathogenic strains of E. coli can cause infections in immunosuppressed or debilitated animals (Nataro and Kaper, 1998).…”

Absolute quantification of E. coli virulence and housekeeping genes to determine pathogen loads in enumerated environmental samples

“…Quantitative real-time PCR (q-PCR) have been used to detect diarrheagenic E . coli (DEC) types by targeting specific virulence genes [ 9 ] using either fluorescent dyes (SYBR ® Green) that bind to double-stranded DNA or fluorescent labelled sequence-specific probes such as Förster resonance energy transfer (FRET) probes; Molecular beacons (PNA based, light probes); Scorpions (duplex); Locked Nucleic Acid (LNA) probes and TaqMan ® probes [ 10 – 12 ].…”