A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters

Abstract

a b s t r a c tThe presence of Escherichia coli in environmental waters is considered as evidence of faecal contamination and is therefore commonly used as an indicator in both water quality and food safety analysis. The long period of time between sample collection and obtaining results from existing culture based methods means that contamination events may already impact public health by the time they are detected. The adoption of molecular based methods for E. coli could significantly reduce the time to detection. A new quantitative real-time PCR (qPCR) assay was developed to detect the ybbW gene sequence, which was found to be 100% exclusive and inclusive (specific and sensitive) for E. coli and directly compared for its ability to quantify E. coli in environmental waters against colony counts, quantitative real-time NASBA (qNASBA) targeting clpB and qPCR targeting uidA. Of the 87 E. coli strains tested, 100% were found to be ybbW positive, 94.2% were culture positive, 100% were clpB positive and 98.9% were uidA positive. The qPCR assays had a linear range of quantification over several orders of magnitude, and had high amplification efficiencies when using single isolates as a template. This compared favourably with qNASBA which showed poor linearity and amplification efficiency. When the assays were applied to environmental water samples, qNASBA was unable to reliably quantify E. coli while both qPCR assays were capable of predicting E. coli concentrations in environmental waters. This study highlights the inability of qNASBA targeting mRNA to quantify E. coli in environmental waters, and presents the first E. coli qPCR assay with 100% target exclusivity. The application of a highly exclusive and inclusive qPCR assay has the potential to allow water quality managers to reliably and rapidly detect and quantify E. coli and therefore take appropriate measures to reduce the risk to public health posed by faecal contamination.Crown

Microbial community dynamics of surface water in British Columbia, Canada

“…(Maheux et al 2011), this qPCR assay detects DNA from viable, viable but non-culturable, as well as dead cells. When compared to other studies (Noble et al 2010; Oliver et al 2016), this association seems to be low, however a drop in correlation coefficients have been reported in environmental water samples (Walker et al 2017).…”

Novel probiotic approach to counter Paenibacillus larvae infection in honey bees

“…All qPCR reactions were performed in DNase-and RNase-free 384-well microplates on a Quant Studio 5 Real-Time PCR System (Applied Biosystems) and analyzed with associated software. Copy numbers of target 16S rRNA genes were calculated as previously described using established primer efficiencies and limit of detections [30][31][32][33].…”

“…Based on observations from the compositional dataset ( Fig. 2c and 3a), Escherichia coli was quantified via qPCR using species-specific primers [33]. Absolute abundance of E. coli in adult nurses was found to be significantly higher on day 12 compared to day 0 for NTC and vehicle-supplemented groups but not the BioPatty-supplemented group (one-way ANOVA with Benjamini and Hochberg multiple comparisons, P = 0.0351, P = 0.0217, P = 0.7302, respectively; Fig.…”

New strategies for the enumeration of enteric pathogens in water

“…Several qPCR methods are listed in Table for quantification of FIB, E . coli , Enterococci, Bacteriodales, and the pathogens Campylobacter , Salmonella , STEC, and Shigella (Yang et al ., ; Reischer et al ., ; Kobayashi et al ., ; Ishii et al ., ; Oster et al ., ; Truchado et al ., ; Martzy et al ., ; Singh et al ., ; Walker et al ., ). The LOD for these qPCR methods varied between 6 and 150 templates per reaction (average of 32 templates/reaction).…”

“…coli target genes, Walker et al . showed that the LOD for the NASBA method showed the same sensitivity as qPCR; however, the correlation between culture results and NASBA was poor (Walker et al ., ). A LAMP system developed by Lin et al .…”