Establishment And Application Of PCV2 Detection Method Based On CRISPR-Cas12a System

Porcine circovirus 2(PCV2)infection causes multi-systemic wasting syndrome(PMWS)in weaned pigs,thereby leading to huge economic losses to the global pig industry.Since the traditional pathogen detection methods require expensive equipments and professional operators,thereby making them not to be suitable for early detection of viruses.Therefore,a rapid,accurate and timely diagnosis method is essential for the prevention and control of this epidemic disease.CRISPR(clustered regularly interspaced short palindromic repeats)-Cas12a system,a new sharp diagnosis tool which utilizes the trans-cleavage activity of Cas12a protein to cut the ssDNA probe with fluorophores in the reaction,thus making visual detection of target DNA possible,has widely been applied to the nucleic acid detection of viruses and bacteria.In this study,a diagnosis method facilitating the on-site detection of PCV2 was established based on CRISPR-Cas12a system and validated with clinical samples,as demonstrated in the following aspects.1.Following the construction of a recombinant plasmid expressing LbCas12a protein,the conditions for the expression of this protein were optimized,including using Rosetta competent cells,culturing at 25℃for 16 hours,purifying by nickel column with gradient imidazole concentration’s Wash Buffer,eluting the relatively pure LbCas12a protein with Wash Buffer containing 30 m M imidazole.Additionally,the purified protein was further confirmed to be Lbcas12a protein by Western Blot assay.2.Subsequently,the cleavage activity of LbCas12a protein targeting to the Rep-PCR products was verified by the fact that the protein displayed the cis-cleavage activity targeting to the target sequence and the trans-cleavage activity targeting to the ssDNA probe.Following the determination of the optimized conditions,the PCR-CRISPR detection system was established,with a detection limit reaching to 10~2～10~3 copies/μL.3.Finally,to satisfy the requirement of on-site detection without related equipments,a LAMP-CRISPR method was established by combining the loop-mediated isothermal amplification(LAMP)technology with the CRISPR-Cas12a system.After the optimization of LAMP reaction for the best amplification,the sensitivity and specificity of LAMP-CRISPR detection system in detecting PCV2 were validated by a low detection limit of 1～10 copies/μL and no cross-reaction with other six porcine viruses,respectively.Furthermore,LAMP-CRISPR method presented a 100%coincidence rate with q PCR technology in analysing 30 clinical PCV2 infected DNA samples.In summary,we have successfully established two PCV2 detection approaches separately combing PCR or LAMP technology and CRISPR-Cas12a system,i.e.PCR-CRISPR and LAMP-CRISPR in this study.Compared with traditional detection ones,these two methods displayed higher sensitivity and specificity,especially the LAMP-CRISPR method having no requirement for expensive instruments and being able to achieve visual detection.Thus,these established methods provided a novel option for the rapid,on-site detection and early prevention of PCV2.