Establishment Of Detection Method Of Porcine Circovirus-Like Virus Based On CRISPR/Cas12a System

Circular single-stranded DNA(CRESS DNA)viruses are a highly diverse group of small viruses.This kind of virus,which is ubiquitous all over the world,has the characteristics of high infectivity and strong adaptability,and plays a crucial role in maintaining the balance of the global ecosystem.Porcine Circovirus-like Virus(PCLV),a member of CRESS DNA viruses,is a closed,circular,non-enveloped DNA virus with similar structure to Porcine Circovirus.The pathogenesis of Porcine circovirus-like virus has not been studied much at home and abroad.Therefore,the development of a rapid,accurate and simple method for detection of PCLV can achieve the purpose of early detection and corresponding measures,which has certain significance for reducing the breeding cost to a certain extent.The CRISPR/Cas system,which is a kind of adaptive immune system in bacteria and archaea,is a popular research direction in recent years.clustered regularly interspaced short palindromic repeats,CRISPR),a series of repeated sequences with unique spacers of similar size.The uniquely repetitive spacer is a nucleic acid record of foreign pathogen invasion,which is used to counter the attack of foreign mobile genetic elements.This system activates Cas protein to non-specifically cut ssDNA after specific target sequence recognition of crRNA,so as to achieve the purpose of in vitro detection.There is no relevant report on the application of CRISPR/Cas system in the detection of PCLV.Therefore,it is of great significance to develop a method to rapidly diagnose PCLV to reduce the breeding cost.In this study,based on Loop-mediated Isothermal Amplification(LAMP),CRISPR/Cas12 a fluorescence and CRISPR/Cas12 a strips were constructed,respectively.This method not only has the characteristics of high sensitivity and specificity,but also has the advantages of simple operation and no need for professional and technical personnel operation,which is of great significance for the early,rapid and efficient detection of PCLV.The test methods and results are as follows:1.Establishment of reaction system and optimization of conditions for CRISPR/Cas12 a fluorescence methodAccording to the conserved sequence of ORF4 gene in PCLV,the recombinant plasmid PCl V-PMD-19 T was constructed.According to the conserved sequence fragment of the constructed recombinant plasmid,three appropriate 5’-TTTN PAM sites were selected as the target of CRISPR/Cas12 a system to detect PCLV.Three pairs of crRNA and a probe were designed.Using the recombinant plasmid PCLV-pMD-19 T as template,the fluorescence values of PCl V-Cr NA-F3 and Pcl V-Cr NA-R3 were the highest.According to the locations of selected primers,three sets of specific LAMP primers were designed using Primer Explorer V5 online website.Using the recombinant plasmid PCLV-pMD-19 T as template,appropriate LAMP primers were screened,which were external primers F3-2 and B3-2,and internal primers FIP-2 and BIP-2,respectively.Using LAMP amplification products as templates,the optimal reaction time of CRISPR/Cas12 a was 30 min,and the optimal reaction temperature was 37℃.The sensitivity test showed that the detection limit of Cris PR/Cas12 A was 1.0×101copies/μL.Specific tests were conducted with PCLV,PCV2,PEDV,CSFV,PRV,PPV and PRRSV as templates and ddH2 O as negative control.The results showed no cross reaction.The results of clinical samples showed that 3 of 36 samples were positive,and the coincidence rate was 100% compared with the results of established TaqMan test method.2.Establishment and condition optimization of CRISPR/Cas12 a test strip detection methodCRISPR/Cas12 a strip method(CRISPR-LF)is based on the CRISPR/Cas12 a system and introduces the chromatographic double antibody sandwich technology.According to the position of CRISPR/Cas12 to detect PCLV,a strip probe was designed.LAMP amplification product was used as the template.The optimal concentration of CRISPR/Cas12 a strip probe(CRISPR-LF)was 5 n M.The sensitivity test showed that the minimum detection limit was1.0×101copies/μL.Specific tests were conducted with PCLV,PCV2,PEDV,CSFV,PRV,PPV and PRRSV as templates and ddH2 O as negative control.The results showed no cross reaction.The results of clinical samples showed that 3 of 36 samples were positive,and the coincidence rate was 100% compared with the results of established TaqMan test method.In this study,based on LAMP,the CRISPR/Cas12 a fluorescence detection system and the CRISPR/Cas12 a lateral cross flow strip signal output method were constructed by primers screening,reaction condition optimization,sensitivity test,specificity test and clinical sample detection.At the same time,TaqMan and PCR technology were used for parallel tests and comparative analysis,and the CRISPR/Cas12 a fluorescence method and CRISPR/Cas12 a strip for rapid detection of PCLV were successfully established.On the basis of independent instruments and professionals,sensitive,specific,rapid and simple detection was realized.For the detection technology in the limited area,it has a good development prospect and has very important practical value in the early diagnosis,prevention and control of PCLV.