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发明授权
CN110567951B 基于CRISPR-Cas12a技术的苹果茎沟病毒可视化检测体系及其检测方法 有权

基于CRISPR-Cas12a技术的苹果茎沟病毒可视化检测体系及其检测方法
摘要：
本发明属于分子生物学领域，涉及基于CRISPR‑Cas12a技术的苹果茎沟病毒可视化检测体系及其检测方法。根据ASGV保守序列设计出crRNA，并制作用于比色检测的AuNP‑DNA，探究Cas12a蛋白体外切割反应时间和linker DNA浓度对检测效果的影响，建立最佳ASGV可视化检测体系及检测方法，该方法为恒温反应条件，并通过CRISPR‑Cas12a‑crRNA复合体识别病毒来提高特异性，且linker DNA具有通用性，不需要昂贵的双标记探针，借助低速离心可在40 min内检测多个样本，其检测下限为5.62×102copies/反应，灵敏度比RT‑PCR高100倍。
摘要（英）：

The invention belongs to the field of molecular biology and relates to an apple stem groove virus visual detection system based on the CRISPR-Cas12a technology and a detection method thereof. CrRNA isdesigned according to an ASGV conserved sequence, AuNP-DNA for colorimetric detection is manufactured, the influence of a Cas12a protein in-vitro cleavage reaction time and linker DNA concentration on a detection effect is explored, and the optimal ASGV visual detection system and the detection method thereof are established. The method is carried out under a constant-temperature reaction condition, a virus is recognized through a CRISPR-Cas12a-crRNA complex to improve the specificity, the linker DNA has universality, expensive double-labeled probes are not needed, a plurality of samples canbe detected within 40 minutes by means of low-speed centrifugation, the lower detection limit is 5.62*102 copies/reaction, and the sensitivity is 100 times higher than that of RT-PCR.

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