Improved detection of Escherichia coli and coliform bacteria by multiplex PCR

Abstract

BackgroundThe presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by β-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified.ResultsIn this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively.ConclusionsThe assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods.

Species specific PCR based detection of Escherichia coli from Indian foods

“…These two sets of primers can be run as multiplex PCR (Data not shown). In recent work Molina et al (2015) have shown that yaiO and lacZ3 primers are unique to E. coli. However, these primers are tested with limited number of samples and not tested in field.…”

Molecular characterization of multi-drug resistant <i>Escherichia coli</i> isolates from tropical environments in Southeast Asia

“…PCR, targeting the uidA gene, was performed to confirm the identities of the E. coli isolates (Bej et al, 1991). The forward and reverse primers with the sequences of 5¢-TGGTAATTACCGACGAAAACGGC-3¢ and 5¢-ACGCGTGGTTACAGTCTTGCG-3¢ (Molina et al, 2015), respectively, were used to specifically detect the presence of the uidA gene with an expected size of 162 bp. The phenotypic antibacterial resistance of each of the selected six E. coli isolates was analyzed using the presence of relevant resistance genes, which are b-lactamase encoding genes (ampC and blaCTX-M), sulfonamide resistance genes (sulI, sulII, and sulIII), folic acid resistance genes (dfrA1 and dfrA5), phosphonic acid resistance genes (fosA, fosA3, and fosC2), phenicol resistance genes (cmlA1, catI, and catII), aminoglycoside resistance genes [aph(3)-Ia, aac(3)-IIa and ant(2≤)-Ia], fluoroquinolone resistance genes (qnrA, qnrB, and qnrS), and TET resistance genes (tetA, tetK, and tetM).…”

Understanding wastewater treatment mechanisms: a review on detection, removal, and purification efficiencies of faecal bacteria indicators across constructed wetlands

“…MultiplexqPCR and quantitative real time (qPCR) are the most useful PCR-based techniques in detecting faecal bacterial pathogens in wastewater (Jason et al, 2014;Diana et al, 2015;Dolapo et al, 2015;Chandra et al, 2016). Multiplex-PCR that uses several oligonucleotide probes to simultaneously detect different microorganisms has increased precision in detecting faecal indicators and/or pathogens in CWs (Bej et al, 1991;Ibekwe et al, 2002;Felipe et al, 2015). To count the microorganisms in CWs, q-PCR has been put into use to quantify the number of target gene copies present in a sample (Hui et al, 2014;Delapo et al, 2015).…”