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commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs\u2019 eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A\/B\/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 \u00b0C with a color-based lateral flow readout. The detection limit of the assay for ALV-A\/B\/J subgroups was 50 copies with no cross reactivity with ALV-C\/D\/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek\u2019s disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A\/B\/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.<\/jats:p>","DOI":"10.3390\/v16071168","type":"journal-article","created":{"date-parts":[[2024,7,22]],"date-time":"2024-07-22T08:24:59Z","timestamp":1721636699000},"page":"1168","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Specific and Sensitive Visual Proviral DNA Detection of Major Pathogenic Avian Leukosis Virus Subgroups Using CRISPR-Associated Nuclease Cas13a"],"prefix":"10.3390","volume":"16","author":[{"given":"Qingqing","family":"Xu","sequence":"first","affiliation":[{"name":"The Pirbright Institute and UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Guildford, Surrey GU24 ONF, UK"},{"name":"UK-China Centre of Excellence for Research on Avian Diseases, Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China"},{"name":"Sino-UK Laboratory for Poultry Disease Research, Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaoyao","family":"Zhang","sequence":"additional","affiliation":[{"name":"The Pirbright Institute and UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Guildford, Surrey GU24 ONF, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yashar","family":"Sadigh","sequence":"additional","affiliation":[{"name":"The Pirbright Institute and UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Guildford, Surrey GU24 ONF, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6422-5901","authenticated-orcid":false,"given":"Na","family":"Tang","sequence":"additional","affiliation":[{"name":"UK-China Centre of Excellence for Research on Avian Diseases, Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China"},{"name":"Sino-UK Laboratory for Poultry Disease Research, Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaqian","family":"Chai","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Shandong Agricultural University, Tai\u2019an 271018, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziqiang","family":"Cheng","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":"Yulong","family":"Gao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, Division of Avian Infectious Diseases, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150008, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5779-273X","authenticated-orcid":false,"given":"Aijian","family":"Qin","sequence":"additional","affiliation":[{"name":"Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou University, Yangzhou 225109, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiqiang","family":"Shen","sequence":"additional","affiliation":[{"name":"UK-China Centre of Excellence for Research on Avian Diseases, Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China"},{"name":"Sino-UK Laboratory for Poultry Disease Research, Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2019-3949","authenticated-orcid":false,"given":"Yongxiu","family":"Yao","sequence":"additional","affiliation":[{"name":"The Pirbright Institute and UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Guildford, Surrey GU24 ONF, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8255-516X","authenticated-orcid":false,"given":"Venugopal","family":"Nair","sequence":"additional","affiliation":[{"name":"The Pirbright Institute and UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Guildford, Surrey GU24 ONF, UK"},{"name":"The Jenner Institute Laboratories, University of Oxford, Oxford OX3 7DQ, UK"},{"name":"Department of Biology, University of Oxford, Oxford OX1 3RB, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,7,20]]},"reference":[{"key":"ref_1","first-page":"11","article-title":"The long view: 40 years of avian leukosis research","volume":"41","author":"Payne","year":"2012","journal-title":"Avian Pathol. 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