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

Abstract

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.

Target‐Enriched Multiplex PCR (Tem‐PCR) Assay for Simultaneous Detection of Salmonella spp., Listeria Monocytogenes and Escherichia Coli O157:H7 in Food

“…Although conventional culture-based method has been generally recognized as the classical method for identifying bacterial pathogens, it is laborious and time-consuming (Karami et al 2011;Park et al 2011). Immunological assays are rapid, but poor specificity with a high false-positive rate.…”

Development of a multiplexed PCR assay combined with propidium monoazide treatment for rapid and accurate detection and identification of three viable Salmonella enterica serovars

“…Several approaches have been used in molecular detection assays for determining cell viability. Commonly, as bacterial mRNA degrades more rapidly than DNA after cell death, mRNA may be more likely to represent viable bacteria (Park et al, 2011). However, extraction of mRNA is technically demanding and RNase can easily degrade mRNA molecules, leading to false-negative results (Alifano, Bruni, & Carlomagno, 1994).…”

“…A major drawback of using enriched culture is the impossibility to detect viable-but-non-culturable (VBNC) cells (Wang, Li, & Mustapha, 2009). To overcome the limitations of culture-based methods, molecular biology-and immunology-based techniques, such as PCR (Levy et al, 2008;Pan & Liu, 2002;Park et al, 2011), quantitative real-time PCR (qRT-PCR) (Bhagwat, 2003;Yang, Qu, Wimbrow, Jiang, & Sun, 2007), loop-mediated isothermal amplification (LAMP) (Chen et al, 2011;Hara-Kudo, Yoshino, Kojima, & Ikedo, 2005;Niessen & Vogel, 2010) and enzyme-linked immunosorbent assay (ELISA) (Jain et al, 2011) have been extensively applied. These methods are easy to perform and have the potential for high throughput application.…”

Members of the family Campylobacteraceae: Campylobacter jejuni, Campylobacter coli

“…The use of PCR after enrichment may reduce the presence of false negative analysis results in cases where the Campylobacter colonies are overgrown by the growth of non-target bacteria on a solid agar medium. Ideally quantitative real-time PCR for Campylobacter detection should been applied directly to water samples (Nogva et al, 2000;Yang et al, 2003;Nam et al, 2005;VanDyke et al, 2010;Clark et al, 2011;Park et al, 2011). These studies have utilized Campylobacter PCR assays targeting the 16S rRNA gene (Bang et al, 2002;Moreno et al, 2003;Josefsen et al, 2004;Hellein et al, 2011), the 23S rRNA gene (Engvall et al, 2002), 16S-23S rDNA internal transcribed (ITS) region (Khan and Edge, 2007) and the flagellin genes flaA and flaB (Waage et al, 1999;Moore et al, 2001).…”