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. coli O157: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. coli O157:H7 when >/=10(3) CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when >/=10(4) 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 >/=10(2) 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 >/=10(4) 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.

FIG. 1

Sensitivity of the fluorogenic 5′ nuclease assay for detecting E. coli O157:H7 in individual MOT (Perkin-Elmer). Tenfold dilutions of E. coli O157:H7 were made in mTSB and mEC in triplicate. One-milliliter aliquots from each dilution were subjected to QIAamp tissue kit DNA recovery, and 5 μl of the recovered DNA solution was PCR amplified with the SZ-I and SZ-II primers in the presence of the SZI-97 fluorogenic probe. Detection and analysis were completed with the ABI Prism sequence detection system. All amplification and detection reactions were completed in MOT. The average ΔRQ values for each dilution were plotted against the average CFU/milliliter as determined by plating each dilution on CT-SMAC. The adjusted ΔRQ threshold value was calculated to be 1.04 (dashed line). Error bars indicate the standard deviation of the mean (n = 6).

FIG. 2

Sensitivity of the fluorogenic 5′ nuclease assay for detecting E. coli O157:H7 in mTSB containing ground beef. Tenfold dilutions of E. coli O157:H7 were made in mTSB in triplicate, and 1.0 ml was added to a tube containing 9.0 ml of ground beef-mTSB mixture (10 g of ground beef, 90 ml of mTSB; incubated for 6 h at 37°C). Aliquots (0.5 ml) were collected for DNA recovery by using the DNA-ER (□) and QIAamp tissue kit (▿) DNA extraction methods, and 5 μl of the recovered DNA solution was amplified with the SZ-I and SZ-II primers in the presence of the SZI-97 fluorogenic probe. Detection and analysis were completed with the ABI Prism sequence detection system. All amplification and detection reactions were completed in MORP. The average ΔRQ values determined from DNA recovered from both DNA extraction methods were plotted against the average CFU/milliliter determined by plating each ground beef dilution on CT-SMAC. The ΔRQ threshold value at 99% confidence limits was calculated to be 0.34. Error bars indicate the standard deviation from the mean (n = 3).