A Rapid and Cost‐Efficient Technique for Simultaneous/Duplex Detection of Listeria Monocytogenes and Escherichia Coli O157:H7 Using Real Time PCR

Abstract

The increasing industrial interest in easy, economical and reliable methods has led to the development and application of DNA-based methods for the detection of microbial pathogens in food. In the present article, we describe the development of a cost-efficient EvaGreen-based real-time Polymerase chain reaction (PCR) technique for simultaneous and practical detection of Listeria monocytogenes (L. monocytogenes) and Escherichia coli O157:H7 (E. coli O157:H7) in the food matrix. EvaGreen-based simplex and duplex real-time PCR showed that specific PCR products were identified by melting curve analysis, and had a reproducible T m of 77.00 6 0.4C for L. monocytogenes and 85.60 6 0.2 for E. coli O157:H7.The absolute detection limit of Eva Green-based simplex and duplex real-time PCR was 0.0001 ng/lL for DNA of both pathogens. The relative detection limit of the simplex and duplex technique was less than 10 cells/mL for the two pathogens. PRACTICAL APPLICATIONSThis study illustrates a rapid, effective and cheap identification method to simultaneously monitor two foodborne pathogens in food samples. The specificity and sensitivity of this method can be used to clearly identify these two foodborne pathogens in practical food samples based on the specific genes of the species. Hence, this detection method is applicable to surveillance measures for these two foodborne pathogens in the food production chain. Moreover, this technique is used by food control laboratories in a secure way.

Invited review: Stress resistance of Cronobacter spp. affecting control of its growth during food production

“…The PCR has been widely used as an initial in vitro DNA amplification method (Stoop et al, 2009;Kaynak and Sakalar, 2016). Many methods using gel electrophoresis to visualize the DNA amplicon have been developed (Table 3; Zhao et al, 2014).…”

Single Locked Nucleic Acid-Enhanced Nanopore Genetic Discrimination of Pathogenic Serotypes and Cancer Driver Mutations

“…g ., nucleic acids amplification-based assays). , E. coli O157:H7 is the most frequently isolated Shiga toxin producing E. coli (STEC) serotype. Because E. coli O157:H7 and non-O157 serotypes only differ by one nucleotide in the uid A gene ( uid A +93), this SNP has been used as a biomarker for distinguishing the O157 pathogenic serotype from all other E. coli serotypes. − The second and third targets are EGFR L858R and KRAD G12D driver mutations. Driver mutations cause malfunctions of proteins, affecting cell proliferation and eventually developing cancers.…”

Single Locked Nucleic Acid-enhanced nanopore genetic discrimination of pathogenic serotypes and cancer driver mutations

“…Because E. coli O157:H7 and non-O157 serotypes only differ by one nucleotide in the uidA gene (uidA +93), this SNP has been used as a biomarker for distinguishing the O157 pathogenic serotype from all other E. coli serotypes. [49][50][51][52][53][54] The second and third targets are EGFR L858R and KRAD G12D driver mutations. Driver mutations cause malfunctions of proteins, affecting cell proliferation and eventually developing cancers.…”