Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. ruysiae in water samples

• Published in: Journal of microbiological methods Vol. 220; p. 106909
• Main Authors: Moinet, Marie, Collis, Rose M., Rogers, Lynn, Devane, Megan L., Biggs, Patrick J., Stott, Rebecca, Marshall, Jonathan, Muirhead, Richard, Cookson, Adrian L.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-05-2024
• Subjects: Absolute quantification; Bacteria; Digital PCR; DNA; Escherichia coli; Fecal indicator organisms; Naturalized Escherichia; Quantitation methods; Real-Time Polymerase Chain Reaction - methods; Water microbial quality; Water Quality; Quantitation methods; LOD; Absolute quantification; Fecal indicator organisms; Digital PCR; MPN; NZ; Cq; LOQ; ddPCR; Naturalized Escherichia; bglC; SBA; Water microbial quality; qPCR; eDNA; NTC; lipB; gDNA
• ISSN: 0167-7012; 1872-8359; 1872-8359
• DOI: 10.1016/j.mimet.2024.106909

Escherichia coli are widely used by water quality managers as Fecal Indicator Bacteria, but current quantification methods do not differentiate them from benign, environmental Escherichia species such as E. marmotae (formerly named cryptic clade V) or E. ruysiae (cryptic clades III and IV). Reliable and specific techniques for their identification are required to avoid confounding microbial water quality assessments. To address this, a multiplex droplet digital PCR (ddPCR) assay targeting lipB (E. coli and E. ruysiae) and bglC (E. marmotae) was designed. The ddPCR performance was assessed using in silico analysis; genomic DNA from 40 local, international, and reference strains of target and non-target coliforms; and spiked water samples in a range relevant to water quality managers (1 to 1000 cells/100 mL). Results were compared to an analogous quantitative PCR (qPCR) and the Colilert method. Both PCR assays showed excellent sensitivity with a limit of detection of 0.05 pg/μL and 0.005 pg/μl for ddPCR and qPCR respectively, and of quantification of 0.5 pg/μL of genomic DNA. The ddPCR allowed differentiation and quantification of three Escherichia species per run by amplitude multiplexing and showed a high concordance with concentrations measured by Colilert once proportional bias was accounted for. In silico specificity testing underlined the possibility to further detect and distinguish Escherichia cryptic clade VI. Finally, the applicability of the ddPCR was successfully tested on environmental water samples where E. marmotae and E. ruysiae potentially confound E. coli counts based on the Most Probable Number method, highlighting the utility of this novel ddPCR as an efficient and rapid discriminatory test to improve water quality assessments.
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•Novel droplet digital PCR assay for environmental DNA applications•Ability to differentiate and quantify E. coli and cryptic clades in a single run•Specific quantitation of fecal E. coli in water with high sensitivity and precision•Reduced turnaround time for in-depth microbial water quality assessments