Cas12a-based On-site And Rapid Nucleic Acid Detection Of African Swine Fever

African swine fever（ASF）is an acute,hemorrhagic,and highly contagious infectious disease caused by African swine fever virus（ASFV）,with a mortality rate approaching 100%.Since the first confirmed ASF outbreak in China on 3 August 2018,more than one million pigs were culled in order to halt further spread which made the pig industry suffered a lot.There is no effective treatment or vaccine for it.Thus,fast and accurate diagnosis is one of the important sections of disease prevention and control.RT-PCR has been widely used in ASF detection,but it requires special instrument platform and skilled operator,which limits its application in field detection.Therefore,a simple,fast and sensitive ASFV on-site detection technology is essential for ASF prevention and control.In recent years,the discovery of new Cas subtypes has enabled CRISPR/Cas technology to demonstrate strong application potential in the field of rapid detection.In this study,we utilized the specific enzyme digestion activity of CRISPR/Cas12 and the"collateral cleavage"activity to construct a rapid on-site nucleic acid detection platform based on Cas12,and the rapid on-site detection technology of ASFV nucleic acid is developed based on this.In this study,the genomic sequences of different ASFV subtypes were analyzed to determine the characteristic conserved sequences,and CRISPR/Cas12 specific detection targets were designed based on that.Specific cr RNA-ASFV was prepared by in vitro transcription.Combining with recombinase-aided amplification technology,we established Cas12-based ASF rapid detection fluorescent reporting system and lateral-flow strip reporting system,and performed specificity and sensitivity assessment.The results showed that the detection sensitivity of the Cas12-mediated ASF rapid detection fluorescence reporting system can reach 10 a M(10^-17M),and the detection sensitivity of the corresponding lateral-flow strip reporting system（Cas12-based On-site and Rapid Detection System,CORDS）can reach 10^-15M.The sensitivity has reached the level of the world-class research team,and the detection process could be finished in about 1h.At the same time,the lateral-flow strip detection system has no cross-reaction with 13 other nucleic acids of virus of common swine disease,which showed highly specific.The freeze-drying effectiveness and accelerated stability test of each component of the detection system found that it meets the current industrial application requirements.Therefore,the CRISPR/Cas12-based rapid and on-site detection system of ASFV has greatly improved the ease of operation.In particular,the test strip system does not require special experimental instruments and complicated professional operation skills.The detection results can be observed by the naked eye in about 1h,which provides a good laboratory methodological basis for the development of ASF’s on-site rapid detection kits.At the same time,the platform also helps the development of other pathogenic microorganisms and disease-related molecular detection methods.