Multiplex PCR Assay for Specific, Sensitive and Rapid detection of three food borne pathogens Salmonella sp, Shigella sp and Listeria monocytogenes

Food-borne illnesses caused by pathogenic bacteria still occur at high frequencies in industrialized nations and developing countries. Conventional microbiological analysis using biochemical tests are time consuming (turn around time up to 7 days), less sensitive and non reliable in most of the cases. This time lag inherent in traditional methods can be replaced by rapid, sensitive detection techniques like multiplex PCR. To achieve an effective detection of Salmonella sp, Shigella sp and Listeria monocytogenes in food materials a multiplex PCR method was standardized in this study. Primers were designed targeting pathogen specific DNA sequence in the genes ipaH, hlyA and sefA of Shigella sp, Listeria monocytogenes and Salmonella sp respectively. Specificity of the primers designed was confirmed by uniplex and multiplex PCR with bacteria (30 different species), fungi and yeast. The multiplex PCR aasay was able to detect all three organisms simultaneously in food materials at a detection sensitivity of 30 ng DNA obtained from each of the three bacteria. Further, the results from examination of food samples from vendor using traditional cultivation methods and multiplex PCR assay were comparatively analyzed. With this assay, simultaneously, all the above three food borne pathogens can be reliably detected with in less than 24 h. Introduction The objective of global health is achieved by food safety and the major health risks are because of food borne diseases. Food borne pathogens are necessary to be examined in farms, food industries and markets. There is a need to identify pathogen quickly so that preventive and controlling measures can be taken up quickly. The conventional method for identification involves pre-enrichment, enrichment and finally selective media which are time consuming, tedious, low throughput and invariably mono specific (detecting only one type of pathogen at a time). Depending on type of pathogen it may take at least 5-10 days for identification and selective enumeration of a pathogen. (Echeverria et al. 1991; ISO 1991; USFDA 1998) Rapid and dependable methods are needed for detection and identification of these pathogens. Advances in molecular techniques and tools, particularly PCR have allowed more reliable microbial identification and surveillance. Identifying a pathogen through DNA sequence analysis gives accurate results for biochemically inert species, uncultivable organisms and those having long cultivation periods. (Edwards and Gibbs 1994; Simon 1999; White et al. 1992) Multiplex PCR (m-PCR) is a variant of PCR enabling the simultaneous amplification of many targets of interest in a single reaction using more than one pair of primers thereby conserving reaction components, saving time and minimizing expenses. Multiplex PCR detection of food borne pathogens was earlier published by many authors for different organisms (Zhou et al. 2013; Chen et al. 2012; Jiang et al. 2011) There is various food borne pathogens that have been identified for food borne illness (Vijayalakshmi et al; 2010). Among them listeriosis caused by Listeria monocytogenes, shigellosis (bacillary dysentery) caused by Shigella (4 species) and salmonellosis caused by Salmonella sp are common pathogens responsible for human health havoc during outbreaks. Scant literature is available for identification of these organisms in a single reaction (Garrido et al. 2012; Hendriksen et al. 2011; Susumu kawasaki et al. 2011; Hamid reza tavakoli et al. 2010; Kwai lin thong et al. 2005). Efforts are made in the present study to develop method for multiplex PCR detection of these three organisms in a single reaction. Materials and Methods Reference cultures and cultural conditions Thirty three known bacterial species, two yeast species and one Aspergillus species listed in Table 1 were obtained from culture bank of NCIM Pune and IMTECH, Chandigarh, India. They were cultivated and maintained on nutrient broth unless and otherwise specified. Purity of all cultures was confirmed by their growth on selective media and biochemical characters. Strains of distantly related genera of yeasts and fungi were also tested for contamination and cultivated on media specified for them. Conventional methods Identification of selected three bacterial species from food material was done according to the standard traditional methods (Bacteriological analytical manual, 1992; Compendium of methods for the microbiological examination of foods, 1992; AOAC, 1995) Bacterial Genomic DNA isolation Column based bacterial genomic DNA isolation kit (Cat No 2022) from Bioserve Biotechnologies, India Pvt Ltd was used for extraction of genomic DNA from gram positive and gram negative strains grown overnight in Luria-Bertani