Open AccessMultiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples
Author(s) -
Park Si Hong,
Hanning Irene,
Jarquin Robin,
Moore Philip,
Donoghue Dan J.,
Donoghue Ann M.,
Ricke Steven C.
Publication year - 2011
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2010.02188.x
Subject(s) - campylobacter jejuni , salmonella , campylobacter , escherichia coli , microbiology and biotechnology , biology , campylobacter coli , serotype , bacteria , multiplex , multiplex polymerase chain reaction , polymerase chain reaction , primer (cosmetics) , taqman , chemistry , gene , bioinformatics , biochemistry , organic chemistry , genetics
Three pathogens, Campylobacter , Salmonella , and Shiga‐toxin‐producing Escherichia coli , are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food‐borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. 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 (m‐PCR) using each specific primer pair simultaneously. 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.