Establishment and application of a method for the detection of porcine circovirus type 2 LAMP-CRISPR/Cas12a
This study aimed to establish a rapid,accurate,and user-friendly detection method for porcine circovirus 2(PCV2).Initially,targeting the ORF2 gene of PCV2,four LAMP and five CRISPR-specific primers were designed and the optimal concentrations of components in the LAMP reaction system and CRISPR detection system were optimized.Following validation of specificity,sensitivity,repeatabili-ty,and compliance,the rapid detection method for PCV2,LAMP-CRISPR/Cas12a,was successfully devel-oped.The results demonstrated that the method's minimum detection threshold was close to 1 copy of plasmid DNA,showing significantly higher sensitivity compared to the PCR method commonly used in our laboratory.Under the same conditions,only PCV2 yielded positive results,while all others tested nega-tive,indicating excellent specificity.The method exhibited good repeatability,with an intra-batch coefficient of variation below 5％and an inter-batch coefficient of variation below 10％.It showed high agreement with qPCR and allowed for visual observation of reaction products under blue light.These findings suggest that the established LAMP-CRISPR/Cas12a system is suitable for the rapid detec-tion of PCV2.
porcine circovirus type 2CRISPR/Cas12a detectionloop-mediated isothermal amplification
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