Multiplex PCR for simultaneous identification of E. coli O157:H7, Salmonella spp. and L. monocytogenes in food

Abstract

The rapid detection of pathogens in food is becoming increasingly critical for ensuring the safety of consumers, since the majority of food-borne illnesses and deaths are caused by pathogenic bacteria. Hence, rapid, sensitive, inexpensive and convenient approaches to detect food-borne pathogenic bacteria is essential in controlling food safety. In this study, a multiplex PCR assay for the rapid and simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes was established. The invA, stx and hlyA genes specifically amplified DNA fragments of 284, 404 and 510 bp from Salmonella spp., L. monocytogenes and E. coli O157:H7, respectively. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity of the multiplex PCR were performed by testing different strains. The multiplex PCR assay was able to specifically simultaneously detect ten colony-forming unit/mL of each pathogen in artificially inoculated samples after enrichment for 12 h. The whole process took less than 24 h to complete, indicating that the assay is suitable for reliable and rapid identification of these three food-borne pathogens, which could be suitable in microbial epidemiology investigation.

Simultaneous multiplex Polymerase Chain Reaction detection of Salmonella spp., Escherichia coli O157, Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes and Campylobacter spp.

“…In the recent food poisoning cases (2013 -2016), Salmonella was identified as the causative agent (Food Safety News, 2014;Astro Awani, 2014;Koris, 2016). Salmonellosis, a disease caused by non-typhoidal Salmonella is commonly associated with poultry, eggs, as well as meat, unpasteurized milk and juices, cheese, raw fruits and vegetables, and spices and nuts (Nguyen et al, 2016). Centers for Disease Control and Prevention (CDC) reported about half of the outbreaks of STEC in the United States from 2011 to 2014 were caused by E. coli O157: H7 also known as STEC O157, and contaminated ground beef and raw vegetables as the common food vehicles for infection.…”

“…Centers for Disease Control and Prevention (CDC) reported about half of the outbreaks of STEC in the United States from 2011 to 2014 were caused by E. coli O157: H7 also known as STEC O157, and contaminated ground beef and raw vegetables as the common food vehicles for infection. The low infectious dose of E. coli O157: H7 (10 -100 CFU) is the reason for high number of cases in outbreaks (Settanni and Corsetti, 2007;Lim et al, 2010;Nguyen et al, 2016). In addition, E. coli infection is more common in lower-income countries (WHO, 2017).…”

“…Listeriosis has a high mortality rate in immunocompromised populations, such as HIV/AIDS and chronic disease patients, elderly, pregnant women and unborn children. L. monocytogenes is a concern in the food industry as it often contaminates ready-to-eat food (Nguyen et al, 2016). According to WHO Regional Office for South-East Asia (SEARO) (2012), the incidence of listeriosis varied between 0.1 and 11.3 cases per 1, 000, 000 population per year in different countries.…”

“…The advantages of using mPCR are that the method is sensitive, saves time and effort as well as laboratory cost (Settanni and Corsetti, 2007;Chen et al, 2012). There have been many studies done in the last decade in successfully developing mPCR procedures for simultaneous detection of three different foodborne pathogens (Park et al, 2006;Germini et al, 2009;AlJobori et al, 2016;Nguyen et al, 2016;Trimoulinard et al, 2017). mPCR is a very useful tool which has been used by other researchers to detect five different foodborne pathogens (Chen et al, 2012), distinguish between five closely-related Salmonella spp.…”

Low cost and comprehensive pork detection in processed food products with a different food matrix

“…Multiplex PCR increases efficiency and reliability for simulta-neous analysis of various animal species (Dalmasso et al 2004). In addition, multiplex PCR works on the target simultaneously which can reduce both costs and time (Matsunaga et al 1999;Fajardo et al 2010;Ali et al 2015;Nguyen et al 2016).…”