Detection of the Escherichia coli pathogenic geneeaewith three real-time polymerase chain reaction methods

Abstract

Several real-time polymerase chain reaction (PCR) methods are currently available to rapidly detect the presence of a specific DNA sequence. When used for detection of pathogenic organisms, the turnaround time for PCR-based methods is much lower than for traditional culture techniques. This study compared the sensitivity of three real-time PCR methods when detecting the Escherichia coli pathogenic gene eae to determine which method is most effective in identifying very low levels of the organism. The three methods were used to detect the eae gene over a range of DNA concentrations. The differences in sensitivity were statistically significant (p<0.05), and SYBR Green I PCR was found to have the lowest detection limit of the three; LUX primers had the highest detection limit. Therefore, using a defined DNA concentration for detecting the eae gene, SYBR Green I is the best alternative.

Detection of Escherichia coli O157:H7 in Ground Beef Using Long-Read Sequencing

“…Variability was noted in the number of times specific genes were detected in the samples, and this is likely due to differences in the gene copy number and the presence of nonpathogenic E. coli . The genes eae [ 24 ], fliC [ 25 ], wzx , and wzy [ 26 ] are single-copy genes. Generally, there is only one copy of each stx subtype gene, as well, but more than one copy can be present [ 27 ].…”

Comparison of In-house and Commercial Real-time PCR Systems for the Detection of Enterobacteriaceae and their Evaluation Within an Interlaboratory Study Using Infant Formula Samples

“…However, Mitchell et al (2009) found that the application of LUX™ primers used in the detection of Chlamydophila pneumoniae in clinical specimens displayed a log less sensitivity than their designed TaqMan-based assay. In their comparative analysis using SYBR Green I, TaqMan® probe and LUX™ primers as the detection format, McCrea et al (2007) confirmed that the hairpin structure of the LUX™ primers may improve the specificity of PCR by reducing mispriming and primer-dimer formation. The primers used by Castillo et al (2006) were reported as being suitable for real-time PCR in the detection of Enterobacteriaceae using primers targeting the 16S ribosomal RNA gene.…”

“…LUX™ primers have been designed for use in a number of studies mainly in virology (Aitichou et al 2005;Antal et al 2007;Chen et al 2004;Nordgren et al 2008;Slavov et al 2008). However, some applications in bacteriology have been reported (Balcazar et al 2007;Kunchev et al 2007;McCrea et al 2007;Mitchell et al 2009;Xu et al 2008).…”