Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition,
real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction. Three pathogens, Campylobacter spp., Shiga toxin-producing Escherichia coli (STEC), and Salmonella spp., are leading PF-562271 research buy causes of bacterial gastroenteritis in the United States and worldwide (Shelton et al., 2006; Botteldoorn et al., CX-5461 ic50 2008; D’Souza et al., 2009). Campylobacter spp. have been estimated to affect 2.4 million people annually, causing approximately 124 deaths and costing $1.2–6 billion (Mead et al., 1999; CDC, 2008). Campylobacter spp. are responsible for 17% of all hospitalizations related to illness, and although Campylobacter spp. have a much lower case fatality rate than Salmonella spp. and E. coli O157:H7,
they account for 5% of food-related deaths (Zhao et al., 2001). The Centers for Disease Control estimates that 73 000 cases of E. coli O157 STEC infections occur annually and are transmitted by food or other vehicles (Rangel et al., 2005). The annual cost of this disease is estimated at $405 million in terms of premature death, medical care, and lost productivity. In the United States,
disease caused by an estimated 1.4 million nontyphoidal Salmonella spp. infections (Rabsch et al., 2001) resulted in 1 68 000 visits to physicians, 15 000 hospitalizations, and 580 deaths annually. The Methocarbamol total cost associated with illnesses due to Salmonella spp. infection is estimated at $3 billion annually in the United States (Faúndez et al., 2004). These pathogens can inhabit the gastrointestinal tract of agricultural animals, including cattle, swine, and poultry, as commensals without causing any signs or symptoms of disease in the animals. While inhabiting the gastrointestinal tract, pathogens can be shed into the environment and may subsequently contaminate water sources (Topp et al., 2009). Other animals including wild birds, rodents, reptiles, amphibians, and deer can carry and shed these pathogens into water sources as well (Pasmans et al., 2008; Pickering et al., 2008). Feces from birds and animals, including cattle, contaminated with Campylobacter spp. have been detected in surface water supplies used as drinking water sources (Bopp et al., 2003). In addition, sewage leaks into ground water have led to the contamination of drinking water and outbreaks of Salmonella spp. and Campylobacter spp. gastroenteritis (O’Reilly et al., 2007).