Xinyi Li, Jianfeng Ma, Yi Luo, Dan Zhu, Lianhui Wang, Shao Su
Accurate, ultrasensitive, and fast detection of the African swine fever virus (ASFV) can efficiently prevent its spread and reduce the losses. Herein, an electrochemical biosensor was designed for high-performance detection of ASFV DNA by coupling with CRISPR technology and signal amplification technology. Porous DNA nanoflowers (DNFs) were prepared by rolling circle amplification (RCA), which was preconjugated Cas12a-crRNA complex to improve detection sensitivity, shorten detection time, and simplify detection steps. In the presence of ASFV DNA, the trans-cleavage activity of Cas12a was activated, degrading DNFs into DNA fragments and causing a significant electrochemical signal change. Combined with the CRISPR-Cas12a system, the detection limit of the DNF-amplified biosensor (3.57 aM) is 3 orders of magnitude lower than that of the conventional RCA-amplified biosensor (2.90 fM). Moreover, the designed electrochemical biosensor showed excellent reproducibility, storage stability, and practical analysis ability, suggesting that it has a promising application in the early diagnosis of African swine fever (ASF).