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

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dc.citation.volumeIn Press
dc.contributor.authorMoinet M
dc.contributor.authorCollis RM
dc.contributor.authorRogers L
dc.contributor.authorDevane ML
dc.contributor.authorBiggs PJ
dc.contributor.authorStott R
dc.contributor.authorMarshall J
dc.contributor.authorMuirhead R
dc.contributor.authorCookson AL
dc.coverage.spatialNetherlands
dc.date.accessioned2024-03-11T22:38:56Z
dc.date.accessioned2024-07-25T06:37:28Z
dc.date.available2024-03
dc.date.available2024-03-11T22:38:56Z
dc.date.available2024-07-25T06:37:28Z
dc.date.issued2024-03-01
dc.description.abstractEscherichia 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.
dc.description.confidentialfalse
dc.format.pagination106909-
dc.identifier.author-urlhttps://www.ncbi.nlm.nih.gov/pubmed/38432551
dc.identifier.citationMoinet M, Collis RM, Rogers L, Devane ML, Biggs PJ, Stott R, Marshall J, Muirhead R, Cookson AL. (2024). Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. Ruysiae in water samples.. J Microbiol Methods. In Press. (pp. 106909-).
dc.identifier.doi10.1016/j.mimet.2024.106909
dc.identifier.eissn1872-8359
dc.identifier.elements-typejournal-article
dc.identifier.issn0167-7012
dc.identifier.number106909
dc.identifier.piiS0167-7012(24)00021-6
dc.identifier.urihttps://mro.massey.ac.nz/handle/10179/70564
dc.languageeng
dc.publisherElsevier BV
dc.publisher.urihttps://www.sciencedirect.com/science/article/pii/S0167701224000216
dc.relation.isPartOfJ Microbiol Methods
dc.rights(c) The author/sen
dc.rights.licenseCC BY-NC-NDen
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/deed.enen
dc.subjectAbsolute quantification
dc.subjectDigital PCR
dc.subjectFecal indicator organisms
dc.subjectNaturalized Escherichia
dc.subjectQuantitation methods
dc.subjectWater microbial quality
dc.titleDevelopment of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. Ruysiae in water samples
dc.typeJournal article
pubs.elements-id487045
pubs.organisational-groupOther
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