Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker

Abstract

The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n ‫؍‬ 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 ؋ 10 7 dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources. E scherichia coli O157:H7 is a major food-borne pathogen responsible for a number of outbreaks in animals, poultry, and humans worldwide (1,7,14). It is estimated that E. coli O157:H7 outbreaks infect over 73,500 people annually in the United States (24), and this pathogen has not only become an important food safety concern but also a serious medical and public health problem. In order to effectively handle future outbreaks in a timely manner, it is necessary to have ample availability of sensitive, specific, and reliable methodologies that can be used for the rapid and selective detection of E. coli O157:H7.To date, great efforts have been made to develop appropriate methodologies for the detection of E. coli O157:H7. The traditional culture methods use selective media or chromogenic agar to detect E. coli O157:H7 (23). However, major limitations of this method are that it takes days to obtain the culture while providing little accurate information about the nature of the strain or isolate itself (39). PCR has been widely used for the detection of E. coli O157:H7 from foods and environmental samples. More recently, real-time PCR is gaining popularity for its enhanced sensitivity and specificity and its speedy turnaround time. Numerous real-time PCR-based methods have been reported for rapid and sensitive detection of E. coli O157:H7 (4, 7, 9, 10). One of the major obstacles for the PCR-based methodologies for sensitive and specific detection...

Analysis of yeh Fimbrial Gene Cluster in Escherichia coli O157:H7 in Order to Find a Genetic Marker for this Serotype

“…Our studies of these sequences demonstrated this fact (see below). Recently a study on the expression of Z3276 along with stx2, in different growth phases showed that the expression of Z3276 increased more than 10 times in stationary phase and the pattern of Z3276 expression was similar to that of the stx2 gene [43]. Pathogenicity of bacteria causes by several virulence factors.…”

“…Pathogenicity of bacteria causes by several virulence factors. Production of Stx is the main virulence feature of E. coli O157:H7 but it cannot be solely responsible for full pathogenicity [43]. For instance, the interaction of intimin with its receptor on host cell surface causes the formation of A/E lesions, which are most important for colonization and thus pathogenesis of this bacterium [44].…”

Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method

“…Another issue in the development of molecular detection methods is the ability to distinguish dead from viable cells (31). This is not a crucial evaluation for this method, since a preenrichment step will be used to amplify the E. coli O157 population.…”

Recent and the Latest Developments in Rapid and Efficient Detection of Salmonella in Food and Water

“…PCR/qPCR may rapidly detect presumptive Salmonella directly or after the pre-enrichment stage; ii) high sensitivity and specificity [43, [49][50][51]; and iii) the potential to amplify lower number of organisms and nonculturable bacteria. The disadvantages of PCR assays include: i) a need for thermal cycling instrument and trained personnel; ii) the potential inhibitors in food matrices may hamper PCR amplification [52]; iii) the LOD is approximately10 2 cells/reaction for qPCR.…”