{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T09:24:03Z","timestamp":1770888243999,"version":"3.50.1"},"reference-count":21,"publisher":"Oxford University Press (OUP)","issue":"2","license":[{"start":{"date-parts":[[2026,1,21]],"date-time":"2026-01-21T00:00:00Z","timestamp":1768953600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100012480","name":"Water Research Australia","doi-asserted-by":"publisher","award":["Project 1150"],"award-info":[{"award-number":["Project 1150"]}],"id":[{"id":"10.13039\/100012480","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Collaboration on Sewage Surveillance of SARS-CoV-2"},{"DOI":"10.13039\/100022899","name":"SA Water","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100022899","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,2,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Microbial source tracking (MST) using Bacteroidales markers provides information on faecal contamination in environmental waters. The marker BacUni targets a range of sources, while HF183 is primarily associated with human faeces. Duplex quantitative PCR (qPCR) assays allow simultaneous detection of both markers, though competition between primer\u2013probe sets can affect accuracy. Digital PCR (dPCR) offers absolute quantification and higher sensitivity, but multiplexing can be challenging when marker concentrations differ. This study compared HF183 and BacUni quantification across singleplex and duplex qPCR formats and duplex dPCR. Synthetic standards revealed variability due to gBlock design and probe orientation, whereas environmental samples showed minimal format-dependent effects. Duplex qPCR provided comparable results to singleplex, with minor underestimation relative to dPCR. These small differences likely reflect the use of dPCR-quantified standards for qPCR calibration, rather than using DNA concentration-based calibration methods. Low-copy HF183 samples highlighted dPCR\u2019s superior sensitivity near detection limits. GoTaq qPCR was the most economical option, especially in duplex format, whereas dPCR offered competitive costs for duplexed samples with the advantage of absolute quantification. These findings demonstrate that duplex qPCR assays reliably quantify HF183 and BacUni in environmental waters, with dPCR serving as a robust complementary method for low-abundance or confirmatory analyses.<\/jats:p>","DOI":"10.1093\/lambio\/ovag011","type":"journal-article","created":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T12:27:20Z","timestamp":1768912040000},"source":"Crossref","is-referenced-by-count":0,"title":["Comparative evaluation of duplex and singleplex qPCR assays for detection of human-associated HF183 and general\n                    <i>Bacteroides<\/i>\n                    markers and implications for duplexing in digital PCR"],"prefix":"10.1093","volume":"79","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8605-1439","authenticated-orcid":false,"given":"Melody","family":"Lau","sequence":"first","affiliation":[{"name":"Future Industries Institute, Adelaide University , Adelaide, SA, 5001 ,","place":["Australia"]},{"name":"SA Water , 250 Victoria Square, Adelaide SA 5000 ,","place":["Australia"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9052-4742","authenticated-orcid":false,"given":"Paul Terence","family":"Monis","sequence":"additional","affiliation":[{"name":"SA Water , 250 Victoria Square, Adelaide SA 5000 ,","place":["Australia"]},{"name":"UniSA STEM, Adelaide University , Adelaide, SA 5001 ,","place":["Australia"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandra Ruth","family":"Keegan","sequence":"additional","affiliation":[{"name":"SA Water , 250 Victoria Square, Adelaide SA 5000 ,","place":["Australia"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3301-0470","authenticated-orcid":false,"given":"Barbara","family":"Drigo","sequence":"additional","affiliation":[{"name":"School of Agriculture, Food and Wine, Adelaide University , 31 Hartley Grove, Urrbrae SA 5064 ,","place":["Australia"]},{"name":"Cooperative Research Centre for Solving Antimicrobial resistance in Agribusiness, Food, and Environments (CRC SAAFE) , Adelaide, SA 5001 ,","place":["Australia"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6465-2233","authenticated-orcid":false,"given":"Erica","family":"Donner","sequence":"additional","affiliation":[{"name":"Future Industries Institute, Adelaide University , Adelaide, SA, 5001 ,","place":["Australia"]},{"name":"Cooperative Research Centre for Solving Antimicrobial resistance in Agribusiness, Food, and Environments (CRC SAAFE) , Adelaide, SA 5001 ,","place":["Australia"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6114-0950","authenticated-orcid":false,"given":"Brendon James","family":"King","sequence":"additional","affiliation":[{"name":"SA Water , 250 Victoria Square, Adelaide SA 5000 ,","place":["Australia"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2026,1,21]]},"reference":[{"key":"2026021203420215600_bib1","doi-asserted-by":"publisher","first-page":"231","DOI":"10.3390\/w8060231","article-title":"Current status of marker genes of bacteroides and related taxa for identifying sewage pollution in environmental waters","volume":"8","author":"Ahmed","year":"2016","journal-title":"Water"},{"key":"2026021203420215600_bib2","doi-asserted-by":"publisher","first-page":"4571","DOI":"10.1128\/AEM.66.10.4571-4574.2000","article-title":"A PCR assay to discriminate human and ruminant feces on the basis of host differences in bacteroides-prevotella genes encoding 16S rRNA","volume":"66","author":"Bernhard","year":"2000","journal-title":"Appl Environ Microbiol"},{"key":"2026021203420215600_bib3","doi-asserted-by":"publisher","first-page":"634","DOI":"10.1093\/clinchem\/hvaf043","article-title":"MIQE 2.0: revision of the minimum information for publication of quantitative real-time PCR experiments guidelines","volume":"71","author":"Bustin","year":"2025","journal-title":"Clin Chem"},{"key":"2026021203420215600_bib4","doi-asserted-by":"crossref","first-page":"ovae137","DOI":"10.1093\/lambio\/ovae137","article-title":"Design, validation, and implementation of multiplex digital PCR assays for simultaneous quantification of multiple targets","volume":"78","author":"de\u00a0Korne-Elenbaas","year":"2024","journal-title":"Lett Appl Microbiol"},{"key":"2026021203420215600_bib5","doi-asserted-by":"publisher","first-page":"5695","DOI":"10.1128\/AEM.70.9.5695-5697.2004","article-title":"Rapid estimation of numbers of fecal bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes","volume":"70","author":"Dick","year":"2004","journal-title":"Appl Environ Microbiol"},{"key":"2026021203420215600_bib6","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1111\/1574-6976.12031","article-title":"Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes","volume":"38","author":"Harwood","year":"2014","journal-title":"FEMS Microbiol Rev"},{"key":"2026021203420215600_bib7","doi-asserted-by":"publisher","first-page":"504","DOI":"10.2144\/97233rr01","article-title":"Multiplex PCR: critical parameters and step-by-step protocol","volume":"23","author":"Henegariu","year":"1997","journal-title":"BioTechniques"},{"key":"2026021203420215600_bib8","doi-asserted-by":"crossref","first-page":"1012","DOI":"10.1093\/clinchem\/hvaa125","article-title":"The digital MIQE guidelines update: minimum information for publication of quantitative digital PCR experiments for 2020","volume":"66","author":"Huggett","year":"2020","journal-title":"Clin Chem"},{"key":"2026021203420215600_bib9","doi-asserted-by":"publisher","first-page":"4956","DOI":"10.1016\/j.watres.2009.07.028","article-title":"Identifying human and livestock sources of fecal contamination in Kenya with host-specific Bacteroidales assays","volume":"43","author":"Jenkins","year":"2009","journal-title":"Water Res"},{"key":"2026021203420215600_bib10","volume-title":"Smart Monitoring for microbial risk assessment","author":"King","year":"2022"},{"key":"2026021203420215600_bib11","doi-asserted-by":"publisher","DOI":"10.2760\/243914,JRC125188","volume-title":"Guidance document on multiplex real-time PCR methods","author":"Grohmann","year":"2021"},{"key":"2026021203420215600_bib12","doi-asserted-by":"crossref","DOI":"10.1093\/lambio\/ovad136","article-title":"Comparison of kits for SARS-CoV-2 extraction in liquid and passive samples","volume":"76","author":"Lau","year":"2023","journal-title":"Lett Appl Microbiol"},{"key":"2026021203420215600_bib13","volume-title":"Australian Drinking Water Guidelines Paper 6 National Water Quality Management Strategy","author":"NHMRC and NRMMC","year":"2011"},{"key":"2026021203420215600_bib14","doi-asserted-by":"publisher","first-page":"1809","DOI":"10.3390\/w14111809","article-title":"An overview of microbial source tracking using host-specific genetic markers to identify origins of fecal contamination in different water environments","volume":"14","author":"Paruch","year":"2022","journal-title":"Water"},{"key":"2026021203420215600_bib15","doi-asserted-by":"publisher","first-page":"673306","DOI":"10.3389\/fmicb.2021.673306","article-title":"The use of ribosomal RNA as a microbial source tracking target highlights the assay host-specificity requirement in water quality assessments","volume":"12","author":"Rytk\u00f6nen","year":"2021","journal-title":"Front Microbiol"},{"key":"2026021203420215600_bib16","doi-asserted-by":"publisher","first-page":"3615","DOI":"10.1016\/j.watres.2007.03.028","article-title":"Relationships between Bacteroides 16S rRNA genetic markers and presence of bacterial enteric pathogens and conventional fecal indicators","volume":"41","author":"Savichtcheva","year":"2007","journal-title":"Water Res"},{"key":"2026021203420215600_bib17","doi-asserted-by":"publisher","first-page":"249","DOI":"10.1111\/j.1462-2920.2004.00702.x","article-title":"Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater","volume":"7","author":"Seurinck","year":"2005","journal-title":"Environ Microbiol"},{"key":"2026021203420215600_bib18","doi-asserted-by":"publisher","first-page":"2009","DOI":"10.1007\/s00216-020-02490-2","article-title":"PCR inhibition in qPCR, dPCR and MPS-mechanisms and solutions","volume":"412","author":"Sidstedt","year":"2020","journal-title":"Anal BioanalChem"},{"key":"2026021203420215600_bib19","doi-asserted-by":"publisher","first-page":"155663","DOI":"10.1016\/j.scitotenv.2022.155663","article-title":"Application of digital PCR for public health-related water quality monitoring","volume":"837","author":"Tiwari","year":"2022","journal-title":"Sci Total Environ"},{"key":"2026021203420215600_bib20","doi-asserted-by":"publisher","first-page":"333","DOI":"10.1080\/00288330.2015.1014376","article-title":"A tiered approach for the identification of faecal pollution sources on an Auckland urban beach","volume":"49","author":"Walker","year":"2015","journal-title":"NZ J Mar Freshwater Res"},{"key":"2026021203420215600_bib21","doi-asserted-by":"publisher","first-page":"1732","DOI":"10.2166\/wst.2021.341","article-title":"Quantitative detection of human- and canine-associated Bacteroides genetic markers from an urban coastal lagoon","volume":"84","author":"Yasar","year":"2021","journal-title":"Water Sci Technol"}],"container-title":["Letters in Applied Microbiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/lambio\/advance-article-pdf\/doi\/10.1093\/lambio\/ovag011\/66511295\/ovag011.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/lambio\/article-pdf\/79\/2\/ovag011\/66511295\/ovag011.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/lambio\/article-pdf\/79\/2\/ovag011\/66511295\/ovag011.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T08:43:25Z","timestamp":1770885805000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/lambio\/article\/doi\/10.1093\/lambio\/ovag011\/8435309"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,21]]},"references-count":21,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2026,2,4]]}},"URL":"https:\/\/doi.org\/10.1093\/lambio\/ovag011","relation":{},"ISSN":["1472-765X"],"issn-type":[{"value":"1472-765X","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,2]]},"published":{"date-parts":[[2026,1,21]]},"article-number":"ovag011"}}