Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples

Authors: HONG PARK, SI - University Of Arkansas; HANNING, IRENE - University Of Arkansas; JARQUIN, ROBIN - University Of Arkansas; Moore, Philip; DONOGHUE, DAN - University Of Arkansas; Donoghue, Ann - Annie; RICKE, STEVEN - University Of Arkansas

Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/26/2010
Publication Date: 1/14/2011
Citation: Hong Park, S., Hanning, I., Jarquin, R., Moore Jr., P.A., Donoghue, D., Donoghue, A.M., Ricke, S. 2011. Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples. FEMS Microbiology Letters. 316:7-15.

Interpretive Summary: Three pathogens, Campylobacter, Salmonella, and Shiga toxin producing Escherichia coli (STEC), are leading causes of bacterial gastroenteritis in the United States and worldwide. For example, Campylobacter species are responsible for 17% of all hospitalizations related to illness, and although Campylobacter spp. has a much lower case fatality rate than Salmonella spp. and E. coli O157:H7, they account for 5% of food-related deaths. 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. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water. Considering the large impact that these three pathogens have on the health of humans, it is important to prevent potential illnesses. Given that water can be a source of these pathogens either directly (drinking water) or indirectly (irrigation water), prevention of illnesses could be accomplished by consistent monitoring of water supplies. We developed two types of PCR assays that can detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR using each specific primer pairs simultaneously. 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. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.

Technical Abstract: Three pathogens, Campylobacter, Salmonella, and Shiga toxin producing Escherichia coli (STEC), are leading causes of bacterial gastroenteritis in the United States and worldwide. For example, Campylobacter species are responsible for 17% of all hospitalizations related to illness, and although Campylobacter spp., has a much lower case fatality rate than Salmonella spp., and E. coli O157:H7, they account for 5% of food-related deaths. 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. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water. Considering the large impact that these three pathogens have on the health of humans, it is important to prevent potential illnesses. Given that water can be a source of these pathogens either directly (drinking water) or indirectly (irrigation water), prevention of illnesses could be accomplished by consistent monitoring of water supplies. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR using each specific primer pairs simultaneously. Under optimized multiplex PCR condition, the assay produced a 90-bp product for Campylobacter spp., a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella spp., 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 [(Campylobacter spp. (80.1C), E. coli O157:H7 (83.3C)], and Salmonella 42 spp. (85.9C). Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.