Rapid Colorimetric Point-of-Care Detection of Live Coliform Bacteria Using PMA-LAMP-mAuNP with Method Validation

Natkamol Thoraneenitiyan; Hathairat Buraphaka; Ilada Choopara; Nutthaphol Khupsathianwong; Kanet Wongravee; Sudaluck Thunyaharn; Wanchai Assavalapsakul; Deborah Dean; Sirirat Kokpol; Naraporn Somboonna

doi:10.28991/HEF-2026-07-01-08

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Natkamol Thoraneenitiyan Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
Hathairat Buraphaka Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
Ilada Choopara Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
Nutthaphol Khupsathianwong Technopreneurship and Innovation Management, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand https://orcid.org/0009-0006-4889-7875
Kanet Wongravee Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand https://orcid.org/0000-0003-1368-9719
Sudaluck Thunyaharn Faculty of Medical Technology, Nakhonratchasima College, Nakhon Ratchasima 30000, Thailand https://orcid.org/0000-0002-1702-3861
Wanchai Assavalapsakul Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand https://orcid.org/0000-0003-2739-3177
Deborah Dean 6) Departments of Medicine and Pediatrics, University of California San Francisco School of Medicine, San Francisco, CA 94143, USA. 7) Department of Bioengineering, Joint Program, University of California San Francisco and University of California Berkeley, San Francisco, CA 94143, USA. 8) Benioff Center for Microbiome Medicine, University of California San Francisco, San Francisco, CA 94143, United States https://orcid.org/0000-0002-4490-1746
Sirirat Kokpol Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
Naraporn Somboonna
naraporn.s@chula.ac.th
2) Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. 9) Omics Sciences and Bioinformatics Center, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. 10) Microbiome Research Unit for Probiotics in Food and Cosmetics, Chulalongkorn University, Bangkok 10330, Thailand. 11) Multi-Omics for Functional Products in Food, Cosmetics and Animals Research Unit, Chulalongkorn University, Bangkok 10330, Thailand https://orcid.org/0000-0002-7830-3509

Vol. 7 No. 1 (2026): March

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Coliform bacteria are ubiquitous in environments, including in the feces of humans and animals, and comprise diverse species. Their presence in drinking water and food products is prohibited (generally limited to ≤10 coliform counts/g or mL). Traditional culture methods detect viable coliforms but require 5-7 days of cultivation, whereas conventional PCR/qPCR assays shorten detection time (~4–5 h) but are costly (~60–100 USD per species per reaction), are unable to discriminate between live and dead cells, and cannot simultaneously detect multiple coliform genera in a single reaction. This study thereby developed a rapid, multiplex, colorimetric assay for viable coliform detection using propidium monoazide–loop-mediated isothermal amplification with multiplex nanogold probes (PMA-LAMP-mAuNP). The assay enables simultaneous detection of multiple live coliform species within 80 minutes (40 min viable genetic extraction, 40 min genetic amplification-detection) with a limit of detection of 1 colony forming unit (CFU), is low-cost (~4-5 USD/reaction), and requires only a portable temperature controller and a simple halogen light source, making it field- and point-of-care–ready. Our PMA-LAMP-mAuNP demonstrated high specificity and sensitivity across live and dead cells, pure and mixed cultures of Escherichia, Shigella, Klebsiella, Enterobacter and Salmonella, as well as against non-coliform species. Visual color readouts allowed quantitative estimation of coliform concentrations with reliable linear regressions (R²=0.9665-0.9804). Furthermore, the assay achieved 100% sensitivity for total bacteria and Escherichia coli (coliform) detection, comparable to the reference culture method (90.91% sensitivity), with consistent relative trueness, relative limits of detection, and zero false positives. Hence, this PMA-LAMP-mAuNP assay is a validated and field-deployable platform for rapid, multiplexed detection of viable coliform bacteria at the point-of-care.

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Rapid Colorimetric Point-of-Care Detection of Live Coliform Bacteria Using PMA-LAMP-mAuNP with Method Validation

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Rapid Colorimetric Point-of-Care Detection of Live Coliform Bacteria Using PMA-LAMP-mAuNP with Method Validation

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