PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5′ Nuclease (TaqMan) Assay

Abstract

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC)eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5′ nuclease assay system was sufficient to correctly identify all E. coliO157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed,eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coliO157:H7 when ≥103 CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when ≥104 CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥102 CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥104 CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed,eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR

“…Use of an 8-h primary soil enrichment, along with the multiplex PCR approach described here, offers the possibility of sensitive detection of E. coli O157:H7 in soil within one working day. Pathogen detection time in soil and water could further be reduced by use of¯uorogenic probes in PCR reactions (Bassler et al 1995;Oberst et al 1998;Sharma et al 1999). This method avoids the need for agarose gel visualization of postampli-®cation products due to the release of a¯uorogenic reporter dye during DNA polymerization (Lee et al 1993).…”

“…Numerous PCR-based assays for E. coli O157:H7 are reported in the literature (e.g. Meng et al 1997;Nagano et al 1998;Oberst et al 1998;Fratamico et al 2000). A muliplex PCR approach, can further improve the speci®city of a PCR assay to the O157:H7 serotype, overcoming a problem observed using only single gene target PCR formats (e.g.…”

Methods for optimizing DNA extraction before quantifying oral bacterial numbers by real-time PCR

“…Detection of bacteria using real-time PCR provides for a relatively sensitive, accurate and rapid identification and quantification method for small numbers of bacteria (Chen et al, 1997;Desjardin et al, 1998;Oberst et al, 1998;Martin et al, 2002;Byun et al, 2004;Nadkarni et al, 2004). Because enumeration of bacteria using real-time PCR is entirely dependent on the amount of DNA measured, the protocols for quantitative DNA extraction and purification become critical.…”

Multiplex touchdown PCR assay to enhance specificity and sensitivity for concurrent detection of four foodborne pathogens in raw milk

“…The primers for detecting E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica were targeted specifically on the rfbE (This study), hlyA (Wu et al 2004), nuc (Brakstad et al 1992) and invA genes (Hoorfar et al 2000), respectively. The rfbE gene encoding O157 lipopolysaccharide and for E. coli O157: H7 serogroup is unique (Oberst et al 1998), hlyA encoding listeriolysin for phagosomal escape into the host cell's cytosol (Wu et al 2004), the nuc (thermonuclease) gene in S. aureus (Brakstad et al 1992) and invA encoding an invasion protein in S. enterica (Hoorfar et al 2000). The primers were synthesized by Macrogen Company from South Korea.…”

“…Hence, food safety organizations have great concern regarding microbiological contaminants and their consequences in food industry and human health. related infections are due to consumption of contaminated milk, ground beef, water, and dairy products (Oberst et al 1998). Escherichia coli O157: H7 even in low dosage of 1-100 colony-forming units (CFU) can cause disease (Paton and Paton 1998).…”