© All Rights Reserved *Corresponding author. Email: chaifung_85@yahoo.com International Food Research Journal 19(2): 461-466 (2012) 1,2 Jeshveen, S. S., 1 Chai, L. C., 1* Pui, C. F. and 1 Son, R. 1 Centre of Excellence for Food Safety Research, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan 2 Advanced Medical and Dental Institute, Universiti Sains Malaysia, 13200 Kepala Batas, Pulau Pinang Optimization of multiplex PCR conditions for rapid detection of Escherichia coli O157:H7 virulence genes Abstract: The main source of E. coli 0157:H7 is cattle, but recent studies showed high percentage of outbreaks contributed by contaminated water. The occurrence of E. coli O157:H7 in environmental water samples poses a potential threat to human health. The aim of this study was to establish a protocol for the detection of the pathogen E. coli O157:H7 and E. coli virulence genes (eaeA, rfbE, hly, stx 1 , and stx 2 ) in a multiplex PCR protocol using six speciic primer pairs. The target genes produced species-speciic amplicons at 625 bp, 397 bp, 296 bp, 166 bp, 210 bp and 484 bp for E. coli O157:H7 (liC h7 gene) and virulence genes (eaeA, rfbE, hly, stx 1 , and stx 2 ) respectively. The results obtained show that the established PCR protocol is suitable for a rapid and speciic analysis of the pathogenic E. coli O157:H7 in environmental water samples for the assessment of microbiological risks. Keywords: Escherichia coli O157:H7, multiplex PCR, optimization Introduction E. coli O157:H7 is a part of the enterohemorrhagic group of E. coli (EHEC). This pathogen produces verotoxins that can cause thrombotic thrombocytopenic purpura (TTP), hemorrhagic colitis and hemolytic ureamic syndrome (HUS) (Law, 2000). Hemolytic ureamic syndrome (HUS), a life threatening complication that causes kidney failure, is developed by about 10% of patients, mostly in elderly people and children (Blackall and Marques, 2004). In the year 1982 in US, a hemorrhagic colitis outbreak caused by hamburger consumption resulted in E. coli O157:H7 to be irst recognized as an important human pathogen. Since then, numerous foodborne cases throughout the world for example in countries like Scotland, Japan, Canada and UK have been linked with this pathogen. In addition, E. coli O157:H7 is recognized as one of the most signiicant foodborne pathogen relating public health especially in South Africa, Europe, Japan and US (Hodges and Kimball, 2005). Six genes of E. coli O157:H7 are generally targeted for PCR conirmation, namely rfbE (O157 antigen), eae (intimin), stx 1 (Shiga toxin 1), stx 2 (Shiga toxin 2), hlyA (hemolysin) and liC h7 (lagellar antigen) (Chapman, 2000). E. coli O157:H7 is able to form vero toxins and this virulence factor is encoded by stx 1 and stx 2 genes respectively. The gene eaeA encodes intimin, responsible for adherence of this pathogen to the intestinal lining and causing human illnesses. Meanwhile, hemolysin is encoded by hlyA gene (Boerling et al., 1999), 0157 antigen by rfbE gene and lagellar antigen by liC h7 gene (Felds et al., 1997). Although the primary reservoirs of this pathogen are cattle and meat products, contaminated water has also been responsible for infection. Outbreaks of this pathogen in Japan, US and Europe, have been reportedly caused by contaminated drinking water (Bertrand and Roig, 2007). For humans, a minimal cell count of about 10–100 are suficient to cause serious complications (Keene et al., 1994). E. coli 0157:H7 outbreaks are on the rise, hence it is important to develope a sensitive, rapid, and species-speciic method to identify this pathogen in water and food. Commercial kits are available in the market for detection but is still deemed time consuming as they require long enrichments prior to detect microorganisms. Thus, a sensitive and rapid technique for detection of this pathogen is required. Recently, traditional microbiological culturing techniques are being replaced by polymerase chain reaction (PCR) based techniques for the identiication and detection of E. coli 0157:H7 as it is less laborious and saves signiicant amount of time (Johnson et al., 1995). PCR requires a small amount of DNA unlike the large numbers required for genetic- based molecular diagnostic methods (Feng, 1993). According to Shah et al., (2009), PCR assays are proven speciic and sensitive in detecting microbial pathogens such as E. coli 0157:H7. Several multiplex