Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula

Abstract

Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.

A quantitative prevalence of Escherichia coliO157 in different food samples using real‐time qPCR method

“…We found this method very specific and sensitive for the detection of E. coli O157 in food samples (detection limit of the developed method was TA B L E 1 Population of E. coli O157 in different food samples observed 1 log of CFU/ml for pure broth culture of E. coli O157). Azinheiro et al (2020) developed new assay based on SYBR green melting curve qPCR method to detect three foodborne pathogens including E. coli O157 and other bacterial pathogens simultaneously in spiked out infant formula samples. They found that this method is specific and sensitive and has the detection limit of <1 CFU/25 gr to detect E. coli O157 in the contaminated infant formula samples (Azinheiro et al, 2020).…”

Application of MinION sequencing as a tool for the rapid detection and characterization of Listeria monocytogenes in smoked salmon

“…The DNA for molecular serogrouping (see Materials and methods Section Molecular serogrouping) was extracted by bacterial thermal lysis. To this end, the protocol described by Azinheiro et al was selected (Azinheiro et al, 2020b ). Briefly, 1 mL of a pure L. monocytogenes culture was centrifuged at 16,000 × g for 2 min, the supernatant was discarded, the pellet was rinsed with 1 mL of TE (10 mM Tris-HCl and 1 mM EDTA, pH 8.0), and centrifuged again.…”

Advancement in Salmonella Detection Methods: From Conventional to Electrochemical-Based Sensing Detection

“…These dyes will bind on the DNA groove as the double-strand DNA is amplified, which then increases the fluorescent intensity [ 86 , 87 , 88 ]. qPCR has been widely used for Salmonella detection in various food, poultry, and veterinary products [ 89 , 90 , 91 ]. In the majority of studies, the invasion gene ( invA ) and tetrathionate reductase gene ( ttr ) were the most targeted gene for Salmonella identification using the qPCR method [ 92 ].…”