Ultra-sensitive and point-of-care detection of Capripoxvirus (CaPV) based on loop-mediated amplification (LAMP) and trans-cleavage activity of CRISPR/Cpfl

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/Cpfl 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/Cpfl 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 CRISPRbased lateral flow test. Due to the introduction of CRISPR/Cpfl, the sensitivity of the method is greatly increased, which is of great significance for the early detection of viruses. Through CRISPR/Cpflmediated fluorescence detection, we can detect CaPV as low as 1.47 x 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 x 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. (C) 2021 Elsevier B.V. All rights reserved.