Visual and label-free ASFV and PCV2 detection by CRISPR-Cas12a combined with G-quadruplex

African swine fever (ASF) and postweaning multisystemic wasting syndrome (PMWS) are acute infectious diseases caused by the African swine fever virus (ASFV) and porcine circovirus type 2 (PCV2). At present, there are no effective vaccines for the prevention of ASFV. PMWS, which is harmful to the domestic and even the world pig industry, is difficult to cure and has a high mortality. So, developing simple, inexpensive, and accurate analytical methods to detect and effectively diagnose ASFV and PCV2 can be conducive to avoid ASFV and PCV2 infection. CRISPR has become a potentially rapid diagnostic tool due to recent discoveries of the trans-cleavage properties of CRISPR type V effectors. Herein, we report the visual detection based on CRISPR-Cas12a (cpf1), which is more convenient than fluorescence detection. Through in vitro cleavage target DNA activation, Cas12a can trans-cleavage ssDNA G-quadruplex. TMB/H2O2 and Hemin cannot be catalyzed by cleavaged G-DNA to produce green color products. This protocol is useful for the detection of ASFV and PCV2 with high sensitivity. This method can enable the development of visual and label-free ASFV and PCV2 detection and can be carried out in the field without relying on instruments or power. This method can complete nucleic acid detection at 37 degrees C without using other instruments or energy. Our research has expanded the application of Cas12a and laid the foundation for the field's rapid detection of viral nucleic acid in future.

Automated summary

A visual detection method based on CRISPR-Cas12a has been developed for highly sensitive detection of ASFV and PCV2. This method is simple, convenient, and does not require instruments or power, making it suitable for field use.