Establishment Of A Method To Detect Porcine Pseudorabies Virus Related Genes Using CRISPR/Cas12a System

Caused by Pseudorabies virus(PRV),pseudorabies is an acute,febrile and highly infectious disease that seriously threatens the safety of pig production by contact.The mortality rate of infected young pigs is up to 100%.In late 2011,an outbreak of pseudorabies(PR)in many Bartha-K61 vaccinated farms in China,severely damaging the pig industry at home and abroad,causing irreparable and significant economic losses to the national economy,human health and animal welfare,and productivity.At present,for disease prevention and control,simple and efficient on-site diagnosis is key to disease prevention by inhibiting the spread and prevalence of diseases from the root.Currently,the common and accurate method for nucleic acid detection is real-time fluorescence quantitative PCR,but this technology requires professional operation and strict experimental process,making it hardly usable on-site.Scientists have been looking for nucleic acid technology that can be quickly applied to on-site detection.One of the most important tools is CRISPR/Cas9-based SHERLOCK system invented by Zhang Feng’s team.In this study,we developed an on-site rapid nucleic acid detection system based on specifical target gene cleavage by CRISPR/Cas12,and non-specific cleavage of fluorescent groups.The results of this paper are as follows:1.We screened two relatively stable genetic strains of PRV,PRV-GE and PRV-TK genes,by multiple sequence gene comparison,and synthesized plasmid vectors containing the virus genes.Look for the PAM site on the gene,design sgRNA nearby.Finally,we then designed in-vitro transcribed sgRNA targeting these genes to activate Cas12,which releases the suppression of fluorescent signals,and can activate Cas12a non-specific cutting reporter probe,and the fluorescence group is separated from the quenched group.The fluorophore group performs its fluorescence function,and the result can be clearly observed directly under the action of the fluorescence reader.2.The PRV-GE gene was detected by cas12a system.Through one-way ANOVA,it was found that sgRNA3 was the most efficient,and the PRV-TK gene was found that sgRNA2 was the most efficient.3.After conventional polymerase chain reaction(PCR)amplification,it is combined with cas12a to detect the reaction.The cas12a fluorescence detection reporting system mediated by PCR amplification of PRV-TK gene and PRV-GE gene has high sensitivity and good specificity.4.By combining the transverse flow test strip with cas12a detection technology,the flow test strip can be detected without a fluorescent reader,and the results are more intuitive.5.The established cas12a detection technology can be used for field detection.This technology has strong specificity.Cas12a fluorescence detection without amplification technology can be completed within 20 min,and the corresponding portable test strip can be completed within 1h.It plays a vital role in early prevention and control.