Yu Dan, Xie Zhixun, Zhang Yanfang, Xie Zhiqin, Fan Qing, Luo Sisi, Xie Liji, Li Meng, Zeng Tingting, Zhang Minxiu, Li Xiaofeng, Wei You, Wu Aiqiong, Wan Lijun
GuangXi Key Laboratory of Veterinary Biotechnology, GuangXi Veterinary Research Institute, Nanning, China.
Key Laboratory of China (Guangxi)-ASEAN Cross-Border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Nanning, China.
Virulence. 2025 Dec;16(1):2521012. doi: 10.1080/21505594.2025.2521012. Epub 2025 Jun 22.
(GyG1) and (GyH1) are the second and third most common gyroviruses identified, respectively, after chicken anaemia virus. They were first reported in 2011 and are currently prevalent worldwide. However, limited research on these pathogens and a lack of prevention and control strategies have necessitated the establishment of a rapid diagnostic technique to address new challenges in infectious diseases. Recombinase acid amplification (RAA) combined with CRISPR - Cas12a or CRISPR - Cas13a technology has major advantages for highly sensitive and rapid diagnosis. Specific targets can activate CRISPR-Cas trans-cleavage activity, resulting in non-specific cleavage of single-stranded DNA by the CRISPR - Cas12a complex and RNA cleavage by the CRISPR - Cas13a complex. In this study, for the first time, we combined RAA-based CRISPR - Cas12a and CRISPR - Cas13a systems for simultaneous differential diagnosis of GyG1 and GyH1 infection. The results showed that dual fluorescence channel RAA-based CRISPR - Cas12a/Cas13a technology could detect GyG1 and GyH1 within one hour, with a minimum detection limit of 1.5 copies of the target DNA standard/µL and no cross-reactivity with other avian pathogens. In addition, this method could be used for clinical detection, with the results exhibiting high consistency with those obtained by qPCR. These findings demonstrate that our RAA-based CRISPR - Cas12a/Cas13a dual-channel detection system can detect two different subtypes of gyrovirus in a sample with good specificity and high sensitivity, improving the detection efficiency and providing a new technique for the study of viral infection dynamics.
（GyG1）和（GyH1）分别是继鸡贫血病毒之后第二和第三常见的环病毒。它们于2011年首次报道，目前在全球范围内普遍存在。然而，对这些病原体的研究有限以及缺乏预防和控制策略，因此有必要建立一种快速诊断技术来应对传染病中的新挑战。重组酶酸性扩增（RAA）与CRISPR-Cas12a或CRISPR-Cas13a技术相结合，在高灵敏度和快速诊断方面具有主要优势。特定靶标可激活CRISPR-Cas反式切割活性，导致CRISPR-Cas12a复合物对单链DNA进行非特异性切割，以及CRISPR-Cas13a复合物对RNA进行切割。在本研究中，我们首次将基于RAA的CRISPR-Cas12a和CRISPR-Cas13a系统结合起来，用于同时鉴别诊断GyG1和GyH1感染。结果表明，基于双荧光通道RAA的CRISPR-Cas12a/Cas13a技术可在1小时内检测到GyG1和GyH1，目标DNA标准的最低检测限为1.5拷贝/微升，且与其他禽病原体无交叉反应。此外，该方法可用于临床检测，结果与qPCR获得的结果具有高度一致性。这些发现表明，我们基于RAA的CRISPR-Cas12a/Cas13a双通道检测系统能够以良好的特异性和高灵敏度检测样本中的两种不同亚型的环病毒，提高了检测效率，并为病毒感染动态研究提供了一种新技术。
