{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T11:03:41Z","timestamp":1785323021591,"version":"3.55.0"},"reference-count":13,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T00:00:00Z","timestamp":1785283200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Science Committee of the Ministry of Science","award":["AP19676553"],"award-info":[{"award-number":["AP19676553"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Avian influenza A virus (AIV) and Newcastle disease virus (NDV) are among the most damaging pathogens of poultry, and AIV also carries zoonotic potential that places it within the One Health agenda. The two infections frequently produce overlapping clinical signs, so a method that detects both in a single reaction has clear practical value for surveillance. We developed a duplex one-step real-time reverse-transcription PCR (RT-qPCR) that detects AIV in the FAM channel and NDV in the ROX channel. Primers and hydrolysis probes were designed against conserved regions of the AIV nucleoprotein and matrix genes and the NDV matrix, phosphoprotein and nucleoprotein genes, after alignment of 500 sequences per virus retrieved from GenBank from different geographic regions. Annealing temperature, primer and probe concentrations were optimised, and recombinant plasmids carrying the target fragments served both as positive controls and as quantitative standards. The assay was specific for AIV and NDV, with no cross-reactivity with infectious bronchitis virus and no signal crossover between channels; its analytical sensitivity reached approximately 10\u201320 RNA copies per reaction. Qualitative results on reference isolates were fully concordant with two commercial veterinary kits (Cohen\u2019s \u03ba = 1.00), and the assay passed inter-laboratory commission testing. This study establishes the design and core analytical performance of a laboratory-developed duplex assay; expanded in silico inclusivity on contemporary sequences, a broader specificity panel, inhibitor-containing matrices, an internal amplification control, and prospective clinical validation are required before routine surveillance deployment.<\/jats:p>","DOI":"10.3390\/pathogens15080802","type":"journal-article","created":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T10:13:47Z","timestamp":1785320027000},"page":"802","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Analytical Validation of a Multiplex Real-Time RT-PCR Assay for Simultaneous Detection of Avian Influenza A Virus and Newcastle Disease Virus"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4701-1992","authenticated-orcid":false,"given":"Yerbol","family":"Burashev","sequence":"first","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7770-8427","authenticated-orcid":false,"given":"Saken","family":"Khaidarov","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Medical Genetics, Kazakh National Medical University named after S.D. Asfendiyarov, Zheltoksan 37A Street, Almaty 050012, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nurdos A.","family":"Aubakir","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"},{"name":"Institute of Biotechnology, National Academy of Sciences of the Kyrgyz Republic, Bishkek 720071, Kyrgyzstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zamira D.","family":"Omarova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ali B.","family":"Tulendibayev","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takhmina U.","family":"Argimbayeva","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tangat T.","family":"Yermekbay","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Khairulla B.","family":"Abeuov","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rashida A.","family":"Rystaeva","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kulyaisan T.","family":"Sultankulova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabit K.","family":"Kokanov","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6299-9399","authenticated-orcid":false,"given":"Nurlan S.","family":"Kozhabergenov","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6976-1540","authenticated-orcid":false,"given":"Gaukhar O.","family":"Shynybekova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergazy Sh.","family":"Nurabayev","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4238-5116","authenticated-orcid":false,"given":"Kuandyk","family":"Zhugunissov","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5882-4964","authenticated-orcid":false,"given":"Mukhit B.","family":"Orynbayev","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding QazBioPharm, Ministry of Health Care of the Republic of Kazakhstan, Gvardeiskiy 080409, Kazakhstan"},{"name":"Research and Production Center \u201cMVA Group\u201d, Village Koksay, Karasai District, Almaty 040921, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"113","DOI":"10.20506\/rst.28.1.1869","article-title":"Pathogenesis and pathobiology of avian influenza virus infection in birds","volume":"28","author":"Swayne","year":"2009","journal-title":"Rev. Sci. Tech."},{"key":"ref_2","unstructured":"World Organisation for Animal Health (WOAH) (2023). Avian Influenza (Including Infection with High Pathogenicity Avian Influenza Viruses). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, WOAH."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"443","DOI":"10.20506\/rst.19.2.1231","article-title":"Newcastle disease and other avian paramyxoviruses","volume":"19","author":"Alexander","year":"2000","journal-title":"Rev. Sci. Tech."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Swayne, D.E. (2013). Newcastle disease. Diseases of Poultry, Wiley-Blackwell. [13th ed.].","DOI":"10.1002\/9781119421481"},{"key":"ref_5","unstructured":"World Organisation for Animal Health (WOAH) (2021). Newcastle Disease (Infection with Newcastle Disease Virus). 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