{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T13:51:39Z","timestamp":1791553899428,"version":"4.3.4"},"reference-count":35,"publisher":"American Chemical Society (ACS)","issue":"10","license":[{"start":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T00:00:00Z","timestamp":1790553600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T00:00:00Z","timestamp":1790553600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T00:00:00Z","timestamp":1790553600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/na","name":"Guangxi Natural Science Foundation Project","doi-asserted-by":"publisher","award":["2023GXNSFAA026260"],"award-info":[{"award-number":["2023GXNSFAA026260"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Guangxi Natural Science Foundation Project","doi-asserted-by":"publisher","award":["2025GXNSFAA069363"],"award-info":[{"award-number":["2025GXNSFAA069363"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Avian leukosis virus subgroup J (ALV-J) is a retrovirus that poses a serious threat to the poultry industry. It is prone to genetic variation, leading to the emergence of multiple epidemic strains, which creates substantial challenges for clinical detection. Although the recombinase polymerase amplification (RPA)-CRISPR\/Cas12a rapid detection assay demonstrates high sensitivity and specificity, it lacks broad applicability across multiple viral strains and is susceptible to amplicon contamination. In this study, a mixed crRNA-based CRISPR\/Cas12a detection system was established. Sequence analysis predicts its potential to cover most currently circulating ALV-J variant strains. By optimizing the amount of RPA mix and the buffer conditions, a one-tube RPA-CRISPR\/Cas12a assay was developed, with the lowest tested concentration giving 9\/9 positive reactions determined to be 1 \u00d7 100 copies\/\u03bcL (equivalent to 15 copies\/reaction in a 15 \u03bcL reaction). To simplify the sample-handling procedure and reduce detection time, a one-pot RPA-CRISPR\/Cas12a assay was established by adding heparin sodium, achieving the lowest consistently positive concentration of 1 \u00d7 102 copies\/\u03bcL (1500 copies\/reaction). Furthermore, for lateral flow assay (LFA) that requires exposure to the environment, the concentration of amplicons was reduced by restricting RPA amplification and employing an enhanced Cas12a protein while maintaining a lowest tested concentration of 1 \u00d7 101 copies\/\u03bcL (150 copies\/reaction). Specificity evaluation revealed no cross-reactivity with nontarget avian viruses, yielding a positive signal only for ALV-J. When applied to clinical samples, the one-tube RPA-CRISPR\/Cas12a assay exhibited complete agreement with RT-qPCR in terms of positive detection rates. The multiple CRISPR\/Cas12a-RPA assays developed in this study feature broad strain coverage in principle, high accuracy, and low contamination risk, providing practical tools for rapid ALV-J detection across diverse scenarios.<\/jats:p>","DOI":"10.1021\/acsinfecdis.6c00419","type":"journal-article","created":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T13:02:20Z","timestamp":1790600540000},"page":"3408-3418","source":"Crossref","is-referenced-by-count":0,"title":["RPA-CRISPR\/Cas12a\nAssay for ALV-J with Broad-Spectrum\nDetection and Reduced Residual Amplicon Load"],"prefix":"10.1021","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-2383-3087","authenticated-orcid":true,"given":"Ruiying","family":"Han","sequence":"first","affiliation":[{"name":"Guangxi University , , , ,","place":["Nanning, Guangxi, China, 530004"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ning","family":"Xiao","sequence":"additional","affiliation":[{"name":"Guangxi Academy of Sciences , , ,","place":["Nanning, Guangxi, China, 530007"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhirui","family":"Wu","sequence":"additional","affiliation":[{"name":"Guangxi University , , , ,","place":["Nanning, Guangxi, China, 530004"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiahua","family":"Wang","sequence":"additional","affiliation":[{"name":"Guangxi University , , , ,","place":["Nanning, Guangxi, China, 530004"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoli","family":"Wang","sequence":"additional","affiliation":[{"name":"Guangxi University , , , ,","place":["Nanning, Guangxi, China, 530004"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaochuan","family":"Tang","sequence":"additional","affiliation":[{"name":"Guangxi University , , , ,","place":["Nanning, Guangxi, China, 530004"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,28]]},"reference":[{"key":"2026100909100135200_cit1","doi-asserted-by":"publisher","first-page":"e1514","DOI":"10.1128\/genomeA.01586-14","article-title":"Complete genome sequence\nof an American avian leukosis virus subgroup j isolate that causes\nhemangiomas and myeloid leukosis","volume":"3","author":"Sanandan","year":"2015","journal-title":"Genome Announce."},{"issue":"17","key":"2026100909100135200_cit2","article-title":"The Emergence,\nDiversification,\nand Transmission of Subgroup J Avian Leukosis Virus Reveals that the\nLive Chicken Trade Plays a Critical Role in the Adaption and Endemicity\nof Viruses to the Yellow-Chickens","volume":"96","author":"Qiaomu","year":"2022","journal-title":"J. Virol."},{"issue":"12","key":"2026100909100135200_cit3","doi-asserted-by":"publisher","DOI":"10.3390\/v14122599","article-title":"Current Epidemiology\nand Co-Infections of Avian Immunosuppressive\nand Neoplastic Diseases in Chicken Flocks in Central China","volume":"14","author":"Zheng","year":"2022","journal-title":"Viruses"},{"issue":"22","key":"2026100909100135200_cit4","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.01232-20","article-title":"The bipartite\nsequence motif in the N- and C-terminal of gp85 of Subgroup J avian\nleukosis virus plays a crucial role in receptor-binding and viral\nentry","volume":"94","author":"Yao","year":"2020","journal-title":"J. Virol."},{"key":"2026100909100135200_cit5","doi-asserted-by":"publisher","first-page":"261","DOI":"10.1080\/03079457.2011.560142","article-title":"An ALV-J isolate is responsible for spontaneous haemangiomas in layer\nchickens in China","volume":"40","author":"Zhang","year":"2011","journal-title":"Avian Pathol."},{"key":"2026100909100135200_cit6","doi-asserted-by":"publisher","first-page":"275","DOI":"10.1016\/j.vetmic.2011.03.037","article-title":"Isolation and characterization of emerging subgroup J avian leukosis\nvirus associated with hemangioma in egg-type chickens","volume":"151","author":"Lai","year":"2011","journal-title":"Vet. Microbiol."},{"key":"2026100909100135200_cit7","doi-asserted-by":"publisher","DOI":"10.1016\/j.meegid.2021.104721","article-title":"Molecular\ncharacteristics of subgroup J avian Leukosis virus isolated from yellow\nbreeder chickens in Guangdong, China, during 2016\u20132019","volume":"89","author":"Peng","year":"2021","journal-title":"Infect., Genet. Evol."},{"key":"2026100909100135200_cit8","doi-asserted-by":"publisher","DOI":"10.3390\/vetsci13020152","article-title":"Research Progress on\nPathogenesis and Prevention of Avian Leukosis\nVirus J Subgroup (ALV-J)","volume":"13","author":"Liu","year":"2026","journal-title":"Vet. Sci."},{"key":"2026100909100135200_cit9","doi-asserted-by":"publisher","DOI":"10.1016\/j.aca.2021.338882","article-title":"Signal amplification\nand output of CRISPR\/Cas-based\nbiosensing systems: A review","volume":"1185","author":"Si-Yuan","year":"2021","journal-title":"Anal. Chim. Acta"},{"key":"2026100909100135200_cit10","doi-asserted-by":"publisher","first-page":"436","DOI":"10.1126\/science.aar6245","article-title":"CRISPR-Cas12a\ntarget binding unleashes\nindiscriminate single-stranded DNase activity","volume":"360","author":"Chen","year":"2018","journal-title":"Science"},{"key":"2026100909100135200_cit11","doi-asserted-by":"publisher","first-page":"491","DOI":"10.1038\/s41422-018-0022-x","article-title":"CRISPR-Cas12a\nhas both cis- and trans-cleavage activities on single-stranded\nDNA","volume":"28","author":"Li","year":"2018","journal-title":"Cell Res."},{"key":"2026100909100135200_cit12","doi-asserted-by":"publisher","DOI":"10.1016\/j.diagmicrobio.2024.116252","article-title":"CRISPR-Cas based diagnostic\ntools:\nBringing diagnosis out of labs","volume":"109","author":"Chhipa","year":"2024","journal-title":"Diagn. Microbiol.\nInfect. Dis."},{"key":"2026100909100135200_cit13","doi-asserted-by":"publisher","DOI":"10.1186\/s12917-016-0902-6","article-title":"A deep sequencing reveals significant\ndiversity among dominant variants and evolutionary dynamics of avian\nleukosis viruses in two infectious ecosystems","volume":"12","author":"Meng","year":"2016","journal-title":"BMC Vet. Res."},{"key":"2026100909100135200_cit14","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2017.01168","article-title":"Dynamic Co-evolution\nand Interaction\nof Avian Leukosis Virus Genetic Variants and Host Immune Responses","volume":"8","author":"Dong","year":"2017","journal-title":"Front. Microbiol."},{"key":"2026100909100135200_cit15","doi-asserted-by":"publisher","first-page":"289","DOI":"10.1007\/s10096-004-1100-1","article-title":"False-positive\nresults and contamination\nin nucleic acid amplification assays: suggestions for a prevent and\ndestroy strategy","volume":"23","author":"Borst","year":"2004","journal-title":"Eur. J. Clin. Microbiol. Infect.\nDis."},{"key":"2026100909100135200_cit16","doi-asserted-by":"publisher","first-page":"98","DOI":"10.1016\/j.tibtech.2024.07.007","article-title":"Trends in\ndeveloping one-pot CRISPR diagnostics strategies","volume":"43","author":"Chen","year":"2025","journal-title":"Trends. Biotechnol."},{"key":"2026100909100135200_cit17","doi-asserted-by":"publisher","DOI":"10.1002\/ctd2.70091","article-title":"The rise of\none-pot nucleic acid detection using CRISPR","volume":"5","author":"Shi","year":"2025","journal-title":"Clin. Transl. Discovery"},{"key":"2026100909100135200_cit18","doi-asserted-by":"publisher","first-page":"286","DOI":"10.1038\/s41551-022-00861-x","article-title":"Fast and sensitive detection of SARS-CoV-2\nRNA using suboptimal protospacer\nadjacent motifs for Cas12a","volume":"6","author":"Lu","year":"2022","journal-title":"Nat. Biomed. Eng."},{"issue":"18","key":"2026100909100135200_cit19","doi-asserted-by":"publisher","first-page":"13307","DOI":"10.1021\/acs.analchem.5c06674","article-title":"Sensitive and Robust\nOne-Pot RPA-CRISPR\/Cas12a Assay with Elimination\nof cis-Cleavage","volume":"98","author":"Han","year":"2026","journal-title":"Anal. Chem."},{"issue":"7","key":"2026100909100135200_cit20","doi-asserted-by":"publisher","first-page":"3511","DOI":"10.1021\/acssensors.3c02485","article-title":"Tuning the\nDynamic Reaction Balance of CRISPR\/Cas12a and RPA in One Pot: A Key\nto Switch Nucleic Acid Quantification","volume":"9","author":"Yao","year":"2024","journal-title":"ACS Sens."},{"key":"2026100909100135200_cit21","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-025-56516-3","article-title":"Tunable\ncontrol of Cas12 activity promotes universal and fast one-pot nucleic\nacid detection","volume":"16","author":"Cheng","year":"2025","journal-title":"Nat. Commun."},{"key":"2026100909100135200_cit22","doi-asserted-by":"publisher","DOI":"10.3390\/ijms251910780","article-title":"Detection\nof Avian Leukosis Virus\nSubgroup J (ALV-J) Using RAA and CRISPR-Cas13a Combined with Fluorescence\nand Lateral Flow Assay","volume":"25","author":"Chen","year":"2024","journal-title":"Int. J. Mol. Sci."},{"key":"2026100909100135200_cit23","doi-asserted-by":"publisher","DOI":"10.3389\/fvets.2024.1424238","article-title":"Rapid detection\nof avian leukemia\nvirus using CRISPR\/Cas13a based lateral flow dipstick","volume":"11","author":"Li","year":"2024","journal-title":"Front. Vet. Sci."},{"key":"2026100909100135200_cit24","doi-asserted-by":"publisher","DOI":"10.3390\/v16071168","article-title":"Specific and Sensitive\nVisual Proviral DNA Detection\nof Major Pathogenic Avian Leukosis Virus Subgroups Using CRISPR-Associated\nNuclease Cas13a","volume":"16","author":"Xu","year":"2024","journal-title":"Viruses"},{"issue":"1","key":"2026100909100135200_cit25","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1021\/acssynbio.2c00496","article-title":"CRISPR-Based\nDiagnostics: Challenges and Potential Solutions toward Point-of-Care\nApplications","volume":"12","author":"Ahmed","year":"2022","journal-title":"ACS Synth. Biol."},{"key":"2026100909100135200_cit26","doi-asserted-by":"publisher","first-page":"206","DOI":"10.1021\/acs.analchem.2c04555","article-title":"Signal\nAmplification by the trans-Cleavage\nActivity of CRISPR-Cas Systems: Kinetics and Performance","volume":"95","author":"Feng","year":"2023","journal-title":"Anal. Chem."},{"key":"2026100909100135200_cit27","doi-asserted-by":"publisher","first-page":"513","DOI":"10.1016\/j.jgg.2019.11.002","article-title":"Advances in\ndetecting and reducing off-target effects generated by CRISPR-mediated\ngenome editing","volume":"46","author":"Li","year":"2019","journal-title":"J. Genet. Genomics"},{"key":"2026100909100135200_cit28","article-title":"Base editors: Expanding\nthe types of DNA damage products harnessed for genome editing","volume":"1","author":"Sifeng","year":"2021","journal-title":"Gene and Genome Editing"},{"issue":"11","key":"2026100909100135200_cit29","doi-asserted-by":"publisher","first-page":"4617","DOI":"10.1021\/acs.analchem.1c04332","article-title":"Development of Cas12a-Based\nCell-Free Small-Molecule Biosensors via Allosteric Regulation of CRISPR\nArray Expression","volume":"94","author":"Ahmed","year":"2022","journal-title":"Anal. Chem."},{"key":"2026100909100135200_cit30","doi-asserted-by":"publisher","DOI":"10.1016\/j.bios.2023.115609","article-title":"A universal\nall-in-one RPA-Cas12a strategy with de novo autodesigner and its application\nin on-site ultrasensitive detection of DNA and RNA viruses","volume":"239","author":"Cailing","year":"2023","journal-title":"Biosens. Bioelectron."},{"issue":"210","key":"2026100909100135200_cit31","doi-asserted-by":"publisher","DOI":"10.3791\/66703","article-title":"Point-of-care CRISPR-based Diagnostics\nwith Premixed and Freeze-dried Reagents","volume":"16","author":"Kongkaew","year":"2024","journal-title":"J.\nVisualized Exp."},{"key":"2026100909100135200_cit32","doi-asserted-by":"publisher","DOI":"10.1016\/j.trac.2025.118542","article-title":"Competition and cooperation: A new idea for one-pot detection by\nisothermal amplification coupled with CRISPR","volume":"194","author":"Jiang","year":"2026","journal-title":"TrAC, Trends Anal. Chem."},{"key":"2026100909100135200_cit33","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.01616-25","article-title":"MARCH2 inhibits\navian leukosis virus replication by targeting gp85 for ubiquitination\nand degradation","volume":"99","author":"Chen","year":"2025","journal-title":"J. Virol."},{"key":"2026100909100135200_cit34","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2025.1684812","article-title":"Isolation and molecular characterization of subgroup J avian leukosis\nvirus in native chicken breeds of China during 2022\u20132025","volume":"16","author":"Liu","year":"2025","journal-title":"Front. Microbiol."},{"key":"2026100909100135200_cit35","doi-asserted-by":"publisher","first-page":"504","DOI":"10.1080\/15476286.2018.1504546","article-title":"PAM identification\nby CRISPR-Cas effector complexes: diversified mechanisms and structures","volume":"16","author":"Gleditzsch","year":"2019","journal-title":"RNA Biol."}],"container-title":["ACS Infectious Diseases"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acsinfecdis.6c00419","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/aidcbc\/article-pdf\/12\/10\/3408\/68677533\/acsinfecdis.6c00419.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/aidcbc\/article-pdf\/12\/10\/3408\/68677533\/acsinfecdis.6c00419.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T13:18:49Z","timestamp":1791551929000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/aidcbc\/article\/12\/10\/3408\/5440016\/RPA-CRISPR-Cas12a-Assay-for-ALV-J-with-Broad"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,28]]},"references-count":35,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,9,28]]},"published-print":{"date-parts":[[2026,10,9]]}},"URL":"https:\/\/doi.org\/10.1021\/acsinfecdis.6c00419","relation":{},"ISSN":["2373-8227"],"issn-type":[{"value":"2373-8227","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,28]]}}}