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

Abstract

Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 ؋ 10 ؊2 to 1 ؋

Recent trends in the detection of pathogenic Escherichia coli O157 : H7

“…These often labor intensive, consisting of sampling, separation, amplification and detection stages, taking days or weeks 4,9,10,14 to perform. With the recent advances in droplet microfluidics 18,34,[46][47][48][49] , multi-functional nano-composites [18][19][20][21][22] and software design 35 it is now possible digitize each of these stages and very accurately quantitate the entire process in a fraction of the time.…”

Genetically Engineered Phages: a Review of Advances over the Last Decade

“…Traditional plating techniques not only are laborious but also often fail to detect pathogens present in samples at low levels (170)(171)(172). Other methods, such as antibody-based ones, do not usually perform well for complex samples without enrichment to amplify the bacterial targets (173).…”

Magnetic micro/nanoparticle flocculation-based signal amplification for biosensing

“…coli 0157 is pathogenic and the presence of it in food samples has to be monitored. Conventional immunomagnetic capturing of E. coli from culture media was performed according to International Organization for Standardization E. coli 0157 detection standard 16654:2001 and a similar procedure can be found in Almeida et al 39 Briefly, E. coli samples were incubated for 6 hours at 41.5°C for preenrichment. For immunomagnetic separation, 1 mL sample was mixed with 20 μL of magnetic beads, Captivate 0157 (Lab M) coated with anti-E. coli O157 antibodies for 10 minutes.…”

“…The excessive dye was rinsed off with water to complete the standard simple staining procedure. 37,39 To perform magnetic dipole-dipole interaction, ferromagnetic beads (8 μm) suspended in PBS buffer added to stained bacteria and the platform, including permanent magnets, were placed on the two sides of the droplet on the glass slide, as in Figure 7. The anti-E. coli magnetic beads were attracted to ferromagnetic beads as a result of magnetic dipole-dipole interaction forming an accumulation as:…”

“…For instance, conventional standard E. coli detection procedure includes magnetic separation of E. coli from food samples and culturing them in petri dishes, which takes more than 24 hours and some detection procedures require fluorescent labels. 39 Together with separation step, our method only takes 20-25 minutes and does not need any fluorescent labels.…”