{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T05:01:36Z","timestamp":1780635696320,"version":"3.54.1"},"reference-count":31,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T00:00:00Z","timestamp":1780444800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"WVDL"},{"name":"ODA"},{"name":"Indical Bioscience"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>This method comparison demonstrated equivalent analytical and diagnostic performance of IndiMix JOE with intype IC-RNA as an alternative real-time polymerase chain reaction (RT-PCR) chemistry compared to the National Animal Health Laboratory Network (NAHLN) influenza A virus (IAV) RT-PCR surveillance assay using AgPath-ID One-Step RT-PCR Reagents in avian swabs and tissues and bovine milk. In avian samples, IndiMix JOE with intype IC-RNA using a fast reduced-volume protocol had comparable results to the NAHLN reference RT-PCR with and without intype IC-RNA using standard NAHLN thermocycling conditions. A six-way comparison of RT-PCR chemistries and exogenous internal controls in milk samples illustrated equivalent mean CT values (ANOVA, p = 0.9938). Additional experiments in avian swabs and tissues, bovine milk and semen samples resulted in comparable analytical sensitivity in limits of detection (LOD), linearity (R2 &gt; 0.977), and PCR efficiencies, and no significant differences in mean CT values (ANOVA, p &gt; 0.05). Diagnostic performance showed 100% agreement across field and spiked sample matrices compared to the NAHLN reference method. The study supports the use of IndiMix JOE and intype IC-RNA as an alternative, with practical improvements relevant to surveillance workflows including enhanced testing flexibility, efficiency, outbreak response capacity, and reduced time to results.<\/jats:p>","DOI":"10.3390\/pathogens15060600","type":"journal-article","created":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T07:58:38Z","timestamp":1780473518000},"page":"600","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of IndiMix JOE with Intype IC-RNA as an Alternative to AgPath-ID for Influenza A Virus Detection in Avian and Bovine Samples"],"prefix":"10.3390","volume":"15","author":[{"given":"Andie","family":"Hach","sequence":"first","affiliation":[{"name":"Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin\u2013Madison, Madison, WI 53706, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne","family":"Vandenburg-Carroll","sequence":"additional","affiliation":[{"name":"Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin\u2013Madison, Madison, WI 53706, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8574-2473","authenticated-orcid":false,"given":"Douglas G.","family":"Marthaler","sequence":"additional","affiliation":[{"name":"INDICAL BIOSCIENCE, 04103 Leipzig, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen","family":"Vadia","sequence":"additional","affiliation":[{"name":"Ohio Department of Agriculture, Animal Disease Diagnostic Laboratory, Reynoldsburg, OH 43068, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-9135-7230","authenticated-orcid":false,"given":"Qirui","family":"Zhang","sequence":"additional","affiliation":[{"name":"Ohio Department of Agriculture, Animal Disease Diagnostic Laboratory, Reynoldsburg, OH 43068, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2953-7541","authenticated-orcid":false,"given":"Melanie","family":"Prarat","sequence":"additional","affiliation":[{"name":"Ohio Department of Agriculture, Animal Disease Diagnostic Laboratory, Reynoldsburg, OH 43068, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1546-5401","authenticated-orcid":false,"given":"Ailam","family":"Lim","sequence":"additional","affiliation":[{"name":"Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin\u2013Madison, Madison, WI 53706, USA"},{"name":"Department of Pathobiological Sciences, University of Wisconsin\u2013Madison, Madison, WI 53706, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,3]]},"reference":[{"key":"ref_1","unstructured":"U.S. Department of Agriculture (2026, May 15). Confirmations of Highly Pathogenic Avian Influenza in Commercial and Backyard Flocks. Confirmations of Highly Pathogenic Avian Influenza in Commercial and Backyard Flocks, Available online: www.aphis.usda.gov\/livestock-poultry-disease\/avian\/avian-influenza\/hpai-detections\/commercial-backyard-flocks."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"110602","DOI":"10.1016\/j.virol.2025.110602","article-title":"Highly pathogenic avian influenza A (H5N1) virus outbreaks in South America in 2022\u20132024: A comprehensive review of an ongoing panzootic","volume":"610","author":"Bruno","year":"2025","journal-title":"Virology"},{"key":"ref_3","unstructured":"Pan American Health Organization, and World Health Organization (2026, May 15). Epidemiological Update Avian Influenza A(H5N1) in the Americas Region. Available online: https:\/\/www.paho.org\/en\/documents\/epidemiological-update-avian-influenza-ah5n1-americas-region-11-march-2026."},{"key":"ref_4","unstructured":"CDC (2026, May 15). H5N1 Bird Flu: Current Situation. Avian Influenza (Bird Flu), Available online: www.cdc.gov\/bird-flu\/situation-summary\/index.html."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"5738","DOI":"10.1038\/s41467-025-60407-y","article-title":"Highly pathogenic avian influenza H5N1 clade 2.3.4.4b genotype B3.13 is highly virulent for mice, rapidly causing acute pulmonary and neurologic disease","volume":"16","author":"Tipih","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2451","DOI":"10.3201\/eid2912.230464","article-title":"Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b Infections in Wild Terrestrial Mammals, United States, 2022","volume":"29","author":"Elsmo","year":"2023","journal-title":"Emerg. Infect. Dis."},{"key":"ref_7","unstructured":"U.S. Department of Agriculture (2026, May 15). Detections of Highly Pathogenic Avian Influenza in Mammals. Detections of Highly Pathogenic Avian Influenza in Mammals, Available online: https:\/\/www.aphis.usda.gov\/livestock-poultry-disease\/avian\/avian-influenza\/hpai-detections\/mammals."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"S\u00e1nchez-Rodr\u00edguez, F., Diaz-Gavidia, C., Ru\u00edz, S., and Jimenez Bluhm, P. (2025). Highly pathogenic avian influenza (HPAI) in South America, 2022\u20132025: Temporality, affected species, and the southwards expansion to the Antarctic region. bioRxiv.","DOI":"10.1101\/2025.10.03.680239"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Sanchez-Rojas, I.C., Bonilla-Aldana, D.K., Solarte-Jimenez, C.L., Bonilla-Aldana, J.L., Acosta-Espa\u00f1a, J.D., and Rodriguez-Morales, A.J. (2025). Highly Pathogenic Avian Influenza (H5N1) Clade 2.3.4.4b in Cattle: A Rising One Health Concern. Animals, 15.","DOI":"10.3390\/ani15131963"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"913","DOI":"10.1038\/s41586-024-08166-6","article-title":"Dairy cows inoculated with highly pathogenic avian influenza virus H5N1","volume":"637","author":"Baker","year":"2025","journal-title":"Nature"},{"key":"ref_11","unstructured":"U.S. Department of Agriculture (2026, May 15). HPAI Confirmed Cases in Livestock. HPAI Confirmed Cases in Livestock, Available online: https:\/\/www.aphis.usda.gov\/livestock-poultry-disease\/avian\/avian-influenza\/hpai-detections\/hpai-confirmed-cases-livestock."},{"key":"ref_12","unstructured":"Animal and Plant Health Inspection Service, and U.S. Department of Agriculture (2026, May 15). The Occurrence of Another Highly Pathogenic Avian Influenza (HPAI) Spillover from Wild Birds into Dairy Cattle, Available online: https:\/\/www.aphis.usda.gov\/sites\/default\/files\/dairy-cattle-hpai-tech-brief.pdf."},{"key":"ref_13","unstructured":"American Veterinary Medical Association (2026, May 15). Avian Influenza Virus Type A (H5N1) in U.S. Dairy Cattle. Available online: https:\/\/www.avma.org\/resources-tools\/animal-health-and-welfare\/animal-health\/avian-influenza\/avian-influenza-virus-type-h5n1-us-dairy-cattle."},{"key":"ref_14","unstructured":"Wageningen University and Research (2026, February 21). First Case of Avian Influenza Antibodies in Cow in Europe. Available online: www.wur.nl\/en\/news\/first-case-avian-influenza-antibodies-cow-europe."},{"key":"ref_15","unstructured":"(2026, February 21). Vijf Koeien Met Antistoffen Tegen Vogelgriep in Noardeast-Frysl\u00e2n, Omrop Frysl\u00e2n, Available online: https:\/\/www.omropfryslan.nl\/nl\/nieuws\/18164381\/vijf-koeien-met-antistoffen-tegen-vogelgriep-in-noardeast-fryslan."},{"key":"ref_16","unstructured":"(2026, February 21). Herd Screening Identifies Four Additional Dairy Cows with HPAI (H5N1) Antibodies in Northeast Friesland, Netherlands. Available online: https:\/\/beaconbio.org\/en\/report\/?reportid=77e03415-7b8f-4018-9542-9a4a4505011c&eventid=1ec4fefc-93d8-4f33-ad46-619bcee7ba3b."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"824","DOI":"10.3201\/eid3205.251639","article-title":"Highly Pathogenic Avian Influenza A(H5N1) Virus RNA in Bovine Semen, California, USA, 2024","volume":"32","author":"Lim","year":"2026","journal-title":"Emerg. Infect. Dis."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Zimmerman, A., Vandenburg-Carroll, A., Marthaler, D.G., and Lim, A. (2025). Optimizing Nucleic Acid Extraction from Extended Bovine Semen for Endemic and High-Consequence Pathogens. Animals, 15.","DOI":"10.20944\/preprints202510.0045.v1"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"758","DOI":"10.1177\/1040638719889315","article-title":"Inhibition monitoring in veterinary molecular testing","volume":"32","author":"Yan","year":"2020","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1016\/j.fsigen.2012.06.013","article-title":"PCR inhibitor removal using the NucleoSpin\u00ae DNA Clean-Up XS kit","volume":"7","author":"Faber","year":"2013","journal-title":"Forensic Sci. Int. Genet."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1014","DOI":"10.1111\/j.1365-2672.2012.05384.x","article-title":"PCR inhibitors\u2013occurrence, properties and removal","volume":"113","author":"Schrader","year":"2012","journal-title":"J. Appl. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1109\/EMR.2020.3018420","article-title":"Impacts of COVID-19 on Global Supply Chains: Facts and Perspectives","volume":"48","author":"Xu","year":"2020","journal-title":"IEEE Eng. Manag. Rev."},{"key":"ref_23","unstructured":"Krueger, K., Main, R., and Zimmerman, J. (2026, February 21). ASF and CSF Negative Cohort Study-Partnering to Expand Testing Capacities, Support Further Evaluation and Validation of Four Commercially Available ASF and CSF PCR Assays, and Enhance Preparedness Across the NAHLN. Proceeding Presented at: NAHLN Farm Bill Showcase-2021 NAHLN Farm Bill (FY22) Funded Projects; November 9, 2023; Virtual, Available online: www.aphis.usda.gov\/sites\/default\/files\/farm-bill-showcase-proceedings-2021.pdf."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Vandenburg-Carroll, A., Marthaler, D.G., and Lim, A. (2025). Enhancing Diagnostic Resilience: Evaluation of Extraction Platforms and IndiMag Pathogen Kits for Rapid Animal Disease Detection. Microbiol. Res., 16.","DOI":"10.20944\/preprints202503.0601.v1"},{"key":"ref_25","unstructured":"(2024). Standard Operating Procedure for Real-Time RT-PCR Detection of Influenza A and Avian Paramyxovirus Type-1 (Standard No. NVSL-SOP-0068). Available online: https:\/\/www.aphis.usda.gov\/labs\/approved-testing-labs\/aphis-approved-testing-laboratory-documents?page=1."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3256","DOI":"10.1128\/JCM.40.9.3256-3260.2002","article-title":"Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes","volume":"40","author":"Spackman","year":"2002","journal-title":"J. Clin. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1637\/8911-043009-Reg.1","article-title":"Validation of a Real-Time Reverse Transcriptase\u2013PCR Assay for the Detection of H7 Avian Influenza Virus","volume":"54","author":"Pedersen","year":"2010","journal-title":"Avian Dis."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1373\/clinchem.2008.112797","article-title":"The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments","volume":"55","author":"Bustin","year":"2009","journal-title":"Clin. Chem."},{"key":"ref_29","unstructured":"World Organisation of Animal Health (2026, February 28). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Thirteenth Edition 2024. Available online: https:\/\/www.woah.org\/fileadmin\/Home\/eng\/Health_standards\/tahm\/A_summry.htm."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"001280","DOI":"10.1099\/jmm.0.001280","article-title":"Impact of different SARS-CoV-2 assays on laboratory turnaround time","volume":"70","author":"Marquis","year":"2021","journal-title":"J. Med. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1177\/17571774221148072","article-title":"Reduction in cycle time for a rapid polymerase chain reaction diagnostic test at the point of care","volume":"24","author":"Nanayakkara","year":"2023","journal-title":"J. Infect. Prev."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/6\/600\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T04:25:43Z","timestamp":1780633543000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/6\/600"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,3]]},"references-count":31,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["pathogens15060600"],"URL":"https:\/\/doi.org\/10.3390\/pathogens15060600","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,3]]}}}