{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T17:04:55Z","timestamp":1786122295812,"version":"build-2736575974"},"reference-count":36,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T00:00:00Z","timestamp":1785974400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"State Key Laboratory of Animal Disease Control and Prevention","award":["SKLADCPKF202406"],"award-info":[{"award-number":["SKLADCPKF202406"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Both Chicken anemia virus (CAV) and Gyrovirus galga 1 (GyVg1) belong to the family Anelloviridae and genus Gyrovirus. They are non-enveloped, single-stranded circular DNA viruses that can infect chicken populations, leading to similar clinical symptoms such as growth retardation and immunosuppression, making clinical differentiation challenging. This study established a duplex TaqMan real-time PCR assay to simultaneously detect CAV and GyVg1. Specific primers and probes were developed to target the VP3 gene of CAV (labeled with VIC) and the VP1 gene of GyVg1 (labeled with FAM). The optimization process involved fine-tuning reaction conditions, such as primer\/probe concentrations and annealing temperature, through a systematic matrix approach. The assay demonstrated remarkable linearity under optimized conditions (0.4 \u03bcmol\/L primers, 0.2 \u03bcmol\/L probes, and an annealing temperature of 52 \u00b0C), yielding high R2 values of 0.9973 for CAV and 0.9981 for GyVg1. The method exhibited high specificity, showing no cross-reactivity with other common avian pathogens. The limits of detection were 1.0 \u00d7 100 copies\/\u03bcL for CAV and 1.0 \u00d7 101 copies\/\u03bcL for GyVg1, making it 10-fold more sensitive than conventional PCR. Intra-assay and inter-assay coefficients of variation were below 1.5% and 2.5%, respectively, indicating excellent reproducibility. When tested on 411 clinical samples from Shandong Province, CAV showed a positivity rate of 48.66% (200\/411), GyVg1 18.73% (77\/411), and a co-detection rate of 17.52% (72\/411). Moreover, 25 complete genome sequences of GyVg1 (2376 bp each) were acquired from positive samples. These sequences exhibited a nucleotide identity ranging from 93.6% to 99.3% among the isolates and 92.2% to 99.6% with reference strains. The phylogenetic analysis, which was based on whole-genome sequences, categorized the Shandong isolates into four clusters (I\u2013IV). Notably, the strains were dispersed across these clusters and were intertwined with references from various regions and hosts. This observation suggests the absence of distinct geographic clustering and implies frequent cross-regional and cross-species transmission. In summary, this duplex real-time PCR assay offers a sensitive, specific, and dependable method for concurrently detecting and distinguishing CAV and GyVg1. The study\u2019s findings reveal a significant co-infection rate and genetic variation in GyVg1, underscoring the importance of increased surveillance and deeper exploration of its pathogenicity and transmission patterns.<\/jats:p>","DOI":"10.3390\/v18080859","type":"journal-article","created":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T09:46:38Z","timestamp":1786009598000},"page":"859","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Establishment and Application of a Duplex TaqMan Real-Time PCR Assay for Simultaneous Detection of Chicken Anemia Virus (CAV) and Gyrovirus Galga 1 (GyVg1)"],"prefix":"10.3390","volume":"18","author":[{"given":"Kai-Xuan","family":"Gu","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, Tai\u2019an 271018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7325-1105","authenticated-orcid":false,"given":"Zhi-Hao","family":"Ren","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, Tai\u2019an 271018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao-Long","family":"Sun","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, Tai\u2019an 271018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Li","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Health Management, Shandong Medicine Technician College, Tai\u2019an 271001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wen-Ping","family":"Cui","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, Tai\u2019an 271018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuang","family":"Chang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, Tai\u2019an 271018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Zhao","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, Tai\u2019an 271018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0448-8467","authenticated-orcid":false,"given":"Yu-Long","family":"Gao","sequence":"additional","affiliation":[{"name":"Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi-Xin","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, Tai\u2019an 271018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Zhang, S., Yuan, S., Yan, T., Li, G., Hao, X., Zhou, D., Li, R., Li, Y., and Cheng, Z. 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Sci., 27.","DOI":"10.3390\/ijms27031384"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/8\/859\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T09:17:03Z","timestamp":1786094223000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/8\/859"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,6]]},"references-count":36,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["v18080859"],"URL":"https:\/\/doi.org\/10.3390\/v18080859","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,6]]}}}