A novel multiplex PCR method for the detection of virulence-associated genes of Escherichia coli O157:H7 in food

Abstract

Shiga toxin-producing Escherichia coli O157:H7 (E. coli O157:H7) strains are foodborne infectious agents that cause a number of life-threatening diseases, including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. Hence, various diagnostic methods have been developed so far to detect shiga toxins such as cell culture, ELISA, Rapid Latex Agglutination (RPLA) and hybridization, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. The aim of this study was to develop a complete, rapid and reliable multiplex PCR (mPCR) method by using two pairs of specific primers to detect either the stx1 or the stx2 gene confirms the presence of E.coli O157:H7. The study results show that stx1F/stx1R primers are specific for stx1 and primers stx2F/stx2R are specific for stx2 genes in E. coli O157:H7. The mPCR method with two pairs of primers for amplifying the stx1, stx2 target genes to detect E. coli O157:H7 in food has been set up successfully. Complete method performed well in both types of food matrices with a detection limit of 3 CFU/25 g or mL of food samples. Tests on 180 food samples have shown a specificity value of 93.75 % (95 % confidence interval [CI], 82.83–100), a sensitivity of 100 % (95 % CI, 83.79–99.85 %), and an accuracy of 96.66 % (CI 95 %, 83.41–99.91 %). Interestingly, results indicate that the mPCR performed as well as the traditional culture methods and can reduce the diagnosis time to 2 days. Finally, complete mPCR method was applied to natural samples covering a wide variety of food types proving that the mPCR method was a rapid and reliable screening method for detection of E. coli O157:H7 in food and environmental samples.

Multiplex polymerase chain reaction detection of Shiga toxin genes and antibiotic sensitivity of Escherichia coli O157:H7 isolated from beef meat in Quetta, Pakistan

“…To ensure food safety, rapid and reliable diagnostic tools are essential for detecting E. coli O157:H7 in different food products. Multiplex real-time polymerase chain reaction (PCR) has emerged as the most attractive alternative to culture-based and immunological-based methods for detecting E. coli O157:H7 (Jinneman, Yoshitomi, & Weagant, 2003;Li, Liu, & Wang, 2017;Tabashsum, Nazneen, Ahsan, Bari, & Yasmin, 2016;Van Giau, Nguyen, Nguyen, Le, & Nguyen, 2016;Zhou et al, 2017).…”

Experimentation on artificial inoculation studies for persistence of shiga‐like toxin‐producing Escherichia coli ( E. coli O157) in agricultural soils and vegetables using real‐time PCR

“…However, PCR‐based techniques have the potential to allow for rapid and sensitive detection of foodborne pathogens. In addition, many authors have demonstrated the advantages of using multiplex PCR methods (Elizaquível & Aznar ; Singh, Batish, & Grover, ; Van Giau, Nguyen, Nguyen, Le, and Nguyen (). In this study, rapid, reliable, and specific qPCR method was successfully employed to detect E. coli O157 in food by analyzing the two of the major virulence genes and achieved detection limit of less than 10 3 cells/ml.…”

Development and Evaluation of a Multiplex PCR for Simultaneous Detection of Five Foodborne Pathogens

“…The strains used for specificity testing are listed in Table 1. For the identification of five food-borne pathogens and the sensitivity of the multiplex PCR assay experiments, the following strains were used: three strains E. coli O157:H7, one produces both Stx1 and Stx2 (NLU), and one produces Stx1 only (NIHE), the last one has Stx2 only (HCMUS), all were obtained from previously worked [37,38], S. aureus ATCC6538, S. enterica ATCC 14028, L. monocytogenes A TCC15313, and V. cholera ATCC 17802. All strains were grown in tryptic soy broth (TSB) or brain heart infusion (Merck, Germany) at37°C for 24 h. The simultaneous enrichment broth (SEB), which used for simultaneous enrichment of five pathogenic bacteria in this study, was described previously by Kobayashi et al [39].Then, the culture broth was used for DNA extraction and subjected to the multiplex PCR assay.…”