The Detection Of African Swine Fever Virus Based On CRISPR Technology

African Swine fever(ASF)is an acute and severe infectious disease,which can infect domestic pigs and wild boars of all ages and breeds.Furthmore,the mortality rate approachs100%.ASF has swept the globe since its discovery in 1921.ASF has been popular in China for more than two years and resulted in more than 10 billion dollars in direct economic losses.There is currently no appropriate treatment option and commercial vaccination available for ASF.ASF control strategies primarily depend on fast detection to detect epidemic in time and culling of herds affected by or potentially exposed to the virus for disease prevention and control.Based on this,the development of sensitive,rapid,specific and convenient detection methods is of great significance to the monitoring and control of ASF.Recently,researchers have discovered that CRISPR-Cas13 a has collateral cleavage activity,which means it can cleave single stranded RNAs non-specifically after cleaving target nucleic acids.And it can be used for single nucleotide polymorphism analysis,pathogen detection,drug resistance analysis,and gene mutation detection.Nucleic acid molecules can be detected at very low copy levels based on isothermal amplification and Cas13a-mediated collateral cleavage of a reporter RNA.Based on the above detection technology,this study developed two rapid detection methods,which were based on qPCR and test strips for RPA-CRISPR detection.The fluorescence signal of the assay induced by collateral cleavage of CRISPR-lwCas13 a was detected by a real-time quantitative PCR instrument.The qPCR-based RPA-CRISPR could detect a single copy of ASFV plasmid or genomic DNA,and had higher sensitivity than traditional qPCR methods.The qPCR-based RPA-CRISPR was highly specific to ASFV and had no cross-reactions with other porcine viruses such as PRRSV,CSFV,etc.A total of 52 field samples have been examined.The detection process of qPCR-based RPA-CRISPR took1 h?1.5 h.And the consistene of the positive rate and negative rate between the assay and TaqMan PCR were 100%.Strip based RPA-CRISPR was suitable for on-site testing,which used lateral flow test strips to interpret the results without equipment.It had same level of sensitivity of TaqMan qPCR,reaching 10 copies/?L,and had no cross-reactivity with other swine pathogens.In the examination of field samples,the detection process of qPCR-based RPA-CRISPR took 1.5 h and the agreement of the negative rate and positive rate were 100%and 70% respectively.Overall,RPA-CRISPR is a rapid,sensitive and specific diagnostic method for ASFV,which provides a new idea for the diagnosis of ASFV and shows the application potential for on-site ASFV detection in swine industry and food security.