Abstract：
Capripoxvirus (CaPV) is one of the common skin diseases infecting cattle and sheep which can cause serious economic losses. Establishing ultra-sensitive, rapid, and point-of-care detection of CaPV is particularly important for hindering its spread. Here, we use the principle that CRISPR/Cpf1 can specifically recognize the target DNA and activate its trans-cleavage activity to identify the CaPV product amplified by loop-mediated amplification (LAMP). Under the designed specific primers, a set of LAMP which can amplify CaPV specifically was established and optimized firstly. Then, the CRISPR/Cpf1 was introduced to identify LAMP products. LAMP can be completed at a constant temperature, thus avoiding the use of temperature-variable instruments, making it possible to detect viruses outside the laboratory. To further satisfy the point-of-care detection of CaPV, we introduced a portable fluorometer and CRISPR-based lateral flow test. Due to the introduction of CRISPR/Cpf1, the sensitivity of the method is greatly increased, which is of great significance for the early detection of viruses. Through CRISPR/Cpf1-mediated fluorescence detection, we can detect CaPV as low as 1.47 × 10-3 TCID50 in 50 min, 1000 times more sensitive than quantitative real-time PCR. Through CRISPR-based lateral flow test, we can visually detect CaPV as low as 1.47 × 10-2 TCID50. Besides, this strategy can be used for the primary samples obtained from the cell culture of CaPV after simple ultrasonic disruption, which eliminates the complicated nucleic acid extraction steps required by traditional methods.
摘要：
Capripoxvirus(CaPV)是牛、羊常见的皮肤病之一,可造成严重的经济损失。建立超敏感，快速,而即时检测CaPV对于阻碍其传播尤为重要。这里,我们使用CRISPR/Cpf1可以特异性识别靶DNA并激活其反式切割活性的原理来鉴定通过环介导扩增（LAMP）扩增的CaPV产物。在设计的特异性引物下，首先建立并优化了一组能特异性扩增CaPV的LAMP。然后，引入CRISPR/Cpf1来鉴定LAMP产品。LAMP可以在恒温下完成，从而避免使用变温仪器，使得在实验室外检测病毒成为可能。为了进一步满足CaPV的即时检测，我们介绍了一种便携式荧光计和基于CRISPR的侧流测试。由于CRISPR/Cpf1的引入，该方法的灵敏度大大提高，这对病毒的早期检测具有重要意义。通过CRISPR/Cpf1介导的荧光检测，我们可以在50分钟内检测到低至1.47×10-3TCID50的CaPV，比实时定量PCR灵敏1000倍。通过基于CRISPR的侧向流动测试，我们可以直观地检测到低至1.47×10-2TCID50的CaPV。此外,该策略可用于简单超声破碎后从CaPV细胞培养中获得的原始样品，这消除了传统方法所需的复杂的核酸提取步骤。
