Rapid detection of porcine circovirus type2 using a combination of RAA isothermal amplification and Cas12a nucleic...

Porcine circovirus type 2 (PCV2) induced postweaning multisystemic wasting syndrome (PMWS) in piglets has caused huge economic losses to the swine industry. Rapid clinical detection of PCV2 is crucial for disease clearance. Instead of conventional PCV2 diagnostic methods, isothermal amplification detection has rapidly developed in recent year, of which RAA/RPA-mediated isothermal amplification supplied effective methods in PCV2 detection. But the potential accuracy ratio and requirement of purified DNA limited its application in clinical samples. Recent studies indicate that the Cas12a protein can be used for nucleic acid detection which brings a new perspective to clinical sample testing for disease. Integrating isothermal amplification with Cas12a detection methods to achieve rapid clinical sample testing represents the future trend in rapid diagnostics. In this study, RAA-mediated isothermal amplification and Cas12a-mediated nucleic testing technology were combined for PCV2-infected clinical samples rapid checking. First, Cas12a protein was expressed from E.coli BL21 (DE3) that containing a Cas12a expressed plasmid and purified and then its biofunction was evaluated. Isothermal amplificated primers and crRNAs were designed based on the ORF-2 sequences of PCV2 genome. Subsequently the optimal reaction condition of RAA-Cas12a detection system was assessed by fluorescence intensity detection. Then the detection sensitivity was evaluated and the assays demonstrates this method can detect much lower copies of PCV2 than LAMP-PCR assays. It also exhibits superior specificity over individual PCR, RPA, and LAMP-PCR. Additionally, this study saves a relatively significant time compared to other detection methods, mainly reflected in steps such as DNA extraction. Finally, Clinical sample testing demonstrates that this method maintains over 98% accuracy consistency with PCR detection. In summary, this study established a rapid RAA-Cas12a-based detection method for PCV2 clinical samples. Its high accuracy and operational simplicity make it suitable for future rapid clinical testing of PCV2.