Development of A RPA-CRISPR/Cas12a Based Rapid Visual Detection Assay for Porcine Parvovirus 7

Abstract

Abstract Background Porcine Parvovirus (PPV) are small, enveloped viruses with single stranded genomic DNA. Till now seven genotypes of PPV have been detected worldwide. They are PPV1 to PPV7 with later was first discovered in 2016 in America and then in Asia and European. It has been reported that PPV7 was a co-infector with Porcine Circovirus 2 (PCV2), PCV3 and Porcine reproductive and respiratory syndrome virus (PRRSV). A rapid, sensitive and specific PPV7 detection method that could be applied in poorly equipped laboratory or event in field could be helpful to reveal its distribution and control the spread of this virus. CRISPR/Cas based systems have exhibited outstanding capacities in the detection of pathogenic microorganisms due to the trans-cleavage activities of the Cas proteins.Results Herein, we established a recombinase polymerase amplification (RPA)-CRISPR/Cas12a based rapid viral detection assay for PPV7. Specific RPA primers and five CRISPR RNAs (crRNAs) were designed and synthesized based on the highly conserved region within the NS1 gene of PPV7. The concentration of crRNA and ssDNA were further optimized. Furthermore, we evaluated the sensitivity, specificity, and clinical effectiveness of the RPA-Cas12a based detection assay. The results indicated that this method could be applied for real-time detection. The detection sensitivity of the novel assay was 100 copies/µl, and there were no cross-reactions with other genotypes of PPV, PCV2, PCV3, PRRSV and pseudorabies virus. The RPA-Cas12a based assay could work well in the detection of clinical samples.Conclusions In summary, we developed a visual, sensitive and specific viral diagnostic method based on CRISPR-Cas12a system for PPV7.