‘Ready Mixed’, improved nucleic acid amplification assays for the detection of Escherichia coli DNA and RNA

Abstract

The selective amplification of E. coli nucleic acid sequences could be used for the early warning of faecal contamination in environmental samples. Modified assays for E. coli DNA and RNA markers are presented with improved integrity and performance over existing methods, and demonstrated using 'ready mixed', preserved reagent mixtures.GeneElute PCR Purification Kit (Sigma, UK) and used directly for RNA synthesis using the Hi Scribe T7 RNA Synthesis Kit (NEB, USA) according to the manufacturers recommended protocol. The synthesised RNA was purified using the RNeasy Mini Kit (Qiagen, UK), and contaminating dsDNA was eliminated using RQ1 RNase-Free DNase (Promega, UK) according to the manufacturers recommended protocol. DNA elimination was confirmed by a null Taqbased PCR. The DNA-free RNA was subsequently purified a second time using the RNeasy Clean-up procedure. The mass of RNA was estimated using a BioAnalyser Instrument, and the RNA 6000 Nano Kit (Agilent, UK). This was used to estimate the number of copies based upon the RNA sequence. A series of RNA copy number standards were prepared by diluting the RNA sample in RT-PCR grade water to between 10 and 10 5 copies per microlitre. Standards were prepared from stock RNA solution immediately prior to use.

Recombinase polymerase amplification for fast, selective, DNA‐based detection of faecal indicator Escherichia coli

“…In contrast, 100% of 30 non-E. coli species could not be detected (no detectable sequence amplification) by the RPA method, and these included closely related species including 5 additional members of the Escherichia genus and 3 members of the Shigella genus. The same selectivity results were obtained using the qPCR method, for which our results were in agreement with those reported in earlier work (Walker et al 2017;McQuillan & Wilson 2019), further confirming the ybbW target sequence as highly inclusive of genetic diversity in E. coli, and highly selective for this species.…”

“…The objective was to demonstrate RPA as a 'faster' alternative to an existing qPCRbased method, with equivalent performance in inclusivity of diverse E. coli environmental strains and selectivity for the target species. RPA primers and probe sequences were designed to anneal with a fragment of the E. coli ybbW gene coding sequence, a genetic locus which has already been determined to be both highly conserved within natural E. coli populations, and highly specific to this species (Walker et al 2017;McQuillan & Wilson 2019). Multiple sequence alignment of ybbW gene sequences from diverse E. coli strains was employed to scrutinise the target sequence for potential oligonucleotide (primers and probe) annealing sites, as described in the materials and methods.…”

“…The novel RPA assay was evaluated for both inclusivity and selectivity against a panel of genomic DNA samples, extracted from diverse E. coli strains and a range of non-E. coli bacterial species. For comparison, an existing ybbW-specific qPCR method, first described by Walker et al (Walker et al 2017) and later refined (McQuillan & Wilson 2019), was tested in parallel. The results are shown in Table 2.…”

“…In 'cycles', a high temperature (>90 o C) is applied to destabilise the DNA duplex and then a lower temperature is applied to promote the annealing and extension of oligonucleotide primers on a single-stranded target sequence by a heat-stable DNA polymerase. Sensitive and specific PCR-based detection of E. coli has been demonstrated by amplifying, for example, fragments of the genes uidA (Frahm & Obst 2003;Silkie et al 2008), tuf (Maheux et al 2011), ybbW (Walker et al 2017;McQuillan & Wilson 2019) and clpB (McQuillan & Wilson 2019), and this has been demonstrated to have a better inclusivity and selectivity than culture (Walker et al 2017). However, there are limitations.…”

“…2011), ybbW (Walker et al . 2017; McQuillan and Wilson 2019) and clpB (McQuillan and Wilson 2019), and this has been demonstrated to have a better inclusivity and selectivity than culture (Walker et al . 2017).…”