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Abstract：
Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, 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\' 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 °C 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\'s 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.
摘要：
禽白血病病毒（ALV）包括一组主要与家禽肿瘤疾病相关的禽逆转录病毒，通常被称为禽白血病。根据包络属性属于不同的子组，ALV亚组A,B,和J(ALV-A，ALV-B,和ALV-J）在家禽种群中最普遍。早期识别和清除受感染鸡群中的病毒脱落鸟类对于根除ALV至关重要。因此,的快速发展,准确,简单易用，和成本有效的现场诊断方法对检测ALV亚群非常重要。Cas13a,一种RNA指导的RNA内切核酸酶，可切割靶单链RNA，还表现出对紧密接近的任何旁观者RNA的非特异性核酸内切酶活性。Cas13的独特反式切割活性已被用于包括几种病毒的多种病原体的分子诊断。这里,我们描述了用于特异性检测ALV-A的前病毒DNA的高度敏感的基于Cas13a的分子测试的开发和应用。B,和J子组。原核表达LwaCas13a,通过离子交换和尺寸排阻色谱纯化，结合重组酶聚合酶扩增（RPA）和T7转录，建立了SHERLOCK（特异性高灵敏度酶促报告子解锁）分子检测系统，用于检测ALV-A/B/J亚群的前病毒DNA。这种新颖的方法需要较少的样品输入和较短的周转时间，基于37°C的等温检测和基于颜色的横向流读数。ALV-A/B/J亚组的检测限为50个拷贝，与ALV-C/D/E亚组和其他禽致癌病毒如网状内皮组织病病毒（REV）和马立克病病毒（MDV）无交叉反应。使用本文描述的CRISPR-Cas13a检测ALV-A/B/J核酸的高灵敏度和特异性视觉方法的开发和评估将有助于根除程序中的ALV检测。
