{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:29:00Z","timestamp":1785414540731,"version":"3.56.0"},"reference-count":26,"publisher":"American Society for Microbiology","issue":"4","license":[{"start":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T00:00:00Z","timestamp":1775520000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T00:00:00Z","timestamp":1775520000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,4,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Viability PCR (vPCR) distinguishes viable from nonviable microorganisms by suppressing amplification from structurally compromised cells or virions. This study compared three viability reagents\u2014ethidium monoazide (EMA), propidium monoazide derivative (PMAxx), and platinum (IV) chloride (PtCl\u2084)\u2014for differentiating viable and heat-inactivated\n                      <jats:italic toggle=\"yes\">Staphylococcus aureus<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                      ),\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      (\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      ), human coronavirus OC43 (HCoV-OC43), and Enterovirus A71 (EV-A71) using real-time and digital PCR. EMA and PtCl\u2084 showed limited or inconsistent suppression of dead-cell or dead-virion signals, whereas PMAxx consistently demonstrated superior performance. Under optimal vPCR treatment conditions\u2014100 \u00b5M PMAxx for\n                      <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                      , 25 \u00b5M PMAxx with 0.01% sodium deoxycholate (DC) for\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      , and 200 \u00b5M PMAxx for both HCoV-OC43 and EV-A71\u2014the assays produced clear live\u2013dead discrimination, with the highest \u0394Cq values reaching approximately 12 cycles in the most responsive organism\u2013condition combinations. PMAxx completely suppressed amplification from heat-inactivated\n                      <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                      (10\u2076 CFU\/100 \u00b5L) and EV-A71 (10\u00b2 PFU\/100 \u00b5L) while preserving signals from viable organisms, with concordant results across PCR platforms. Applying these optimized conditions to spiked matrices (milk, vegetable wash water, and nasopharyngeal transport medium) reduced \u0394Cq separation relative to culture suspensions, indicating matrix-associated interference, yet PMAxx maintained effective viability discrimination in most scenarios. This comparative analysis, with digital PCR serving as confirmatory evidence in representative high-contrast conditions, demonstrates that PMAxx provides the most reliable viability discrimination among the reagents evaluated and may be applicable to broader microbial viability studies and environmental surveillance contexts.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Molecular diagnostic assays are indispensable for rapid pathogen detection; however, their inability to distinguish viable from nonviable microorganisms can result in an overestimation of infection risk. The vPCR approach overcomes this limitation by combining selective nucleic acid-intercalating reagents with amplification-based detection. In this study, we evaluated EMA, PMAxx, and PtCl\u2084 across four representative pathogens\u2014\n                        <jats:italic toggle=\"yes\">S<\/jats:italic>\n                        .\n                        <jats:italic toggle=\"yes\">aureus<\/jats:italic>\n                        ,\n                        <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                        , HCoV-OC43, and EV-A71\u2014using both real-time and digital PCR platforms. The results reveal that PMAxx provides superior performance in eliminating false-positive signals from nonviable cells and virions while preserving amplification from viable targets. The incorporation of digital PCR further enhances quantitative accuracy and absolute measurement of pathogen viability. These findings underscore the translational potential of PMAxx-based vPCR as a practical and robust strategy for improving molecular diagnostics, monitoring environmental contamination, and strengthening public health surveillance.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.04002-25","type":"journal-article","created":{"date-parts":[[2026,3,11]],"date-time":"2026-03-11T13:01:28Z","timestamp":1773234088000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Comparative evaluation of viability PCR reagents highlights the superior efficacy of PMAxx azo dye for bacterial and viral viability discrimination using real-time and digital PCR"],"prefix":"10.1128","volume":"14","author":[{"given":"Tiwawan","family":"Hongsibsam","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/002yp7f20","id-type":"ROR","asserted-by":"publisher"}],"name":"Graduate Program in Medical Technology, Faculty of Allied Health Sciences, Thammasat University","place":["Pathum Thani, Thailand"]},{"name":"Department of Medical Technology and Clinical Pathology, Jainad Narendra Hospital","place":["Chainat, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jarunee","family":"Prasertsopon","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01znkr924","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for Research Innovation and Biomedical Informatics, Faculty of Medical Technology, Mahidol University","place":["Nakhon Pathom, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pilaiwan","family":"Siripurkpong","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/002yp7f20","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University","place":["Pathum Thani, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5364-6716","authenticated-orcid":true,"given":"Nattamon","family":"Niyomdecha","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/002yp7f20","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University","place":["Pathum Thani, Thailand"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cll.2022.09.010"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2672.1994.tb03047.x"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1186\/s40168-017-0285-3"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.01763-14"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1111\/jam.12365"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.wroa.2020.100080"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.scitotenv.2022.154258"},{"key":"e_1_3_4_9_2","unstructured":"Bertini I Gray HB Stiefel EI Valentine JS. 2015. 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