Abstract：
Lumpy skin disease virus (LSDV) is a severe and highly contagious form of cowpox. As LSDV continues to mutate and there is no vaccine and treatment in nonendemic countries, early detection of LSDV becomes an important basis for epidemic prevention and control, especially for detection of conserved sequences. A new label-free and sensitive fluorescence method was developed based on a light-up RNA aptamer for detecting LSDV. The method integrated recombinase polymerase amplification (RPA), CRISPR/Cas12a, 10-23 DNAzyme, and Baby Spinach RNA aptamer for triple cascade signal amplification. Based on highly sensitive and specific RPA and CRISPR/Cas12a, DNAzyme achieved a third signal amplification. Additionally, the Baby Spinach RNA aptamer had stronger fluorescence signals and higher quantum yields. The label-free method had ultrahigh sensitivity with the actual detection limit as 1.29 copies·μL-1. The method was 100-fold more sensitive compared to RPA with Cas12a. Moreover, it had no cross-reactivity with viruses belonging to the Capripoxvirus, such as sheep pox virus and goat pox virus with genetic homology as 97%. Furthermore, the method displayed 100% accuracy in 50 actual samples. Therefore, the method based on RPA, Cas12a, and 10-23 DNAzyme had advantages in LSDV detection and provided a new solution for LSD prevention and control.
摘要：
结节性皮肤病病毒（LSDV）是一种严重且高度传染性的牛痘形式。由于LSDV继续变异，并且在非流行国家没有疫苗和治疗，早期发现LSDV成为疫情防控的重要依据,特别是用于检测保守序列。基于发光RNA适体开发了一种新的无标记且灵敏的荧光法，用于检测LSDV。该方法整合重组酶聚合酶扩增(RPA),CRISPR/Cas12a,10-23DNA酶,和婴儿菠菜RNA适体用于三重级联信号扩增。基于高度敏感和特异性的RPA和CRISPR/Cas12a，DNA酶实现了第三个信号放大。此外,婴儿菠菜RNA适体具有更强的荧光信号和更高的量子产率。无标记方法具有超高的灵敏度，实际检测限为1.29拷贝·μL-1。与使用Cas12a的RPA相比，该方法的灵敏度是100倍。此外，它与Capropoxvirus病毒没有交叉反应，如羊痘病毒和羊痘病毒的遗传同源性为97%。此外，该方法在50个实际样品中显示出100%的准确度。因此,基于RPA的方法，Cas12a,10-23DNAzyme在LSDV检测中具有优势，为LSD的预防和控制提供了新的解决方案。
