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 × 100 copies/μL (equivalent to 15 copies/reaction in a 15 μL 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 × 102 copies/μL (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 × 101 copies/μL (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.
