Establishment of a rapid detection method for porcine pavovirus based on PfAgo technology
In order to establish a sensitive,rapid,and specific detection method for porcine parvovirus(PPV)by combining the optimized PfAgo fluorescence method with loop-mediated isothermal amplification(LAMP)technology,gDNA was screened for the conserved sequence of the VP2 gene of PPV,and corresponding molecu-lar beacons were designed to preliminary establish a PfAgo based fluorescence detection method.Primers for LAMP were screened at the same target locus and their amplification conditions were optimized.The combination of the two methods were then investigated for sensitivity and specificity,and the optimized method was ultimately used for the detection of clinical samples.The results showed that the optimal reaction temperature for the PfAgo fluorescence assay was 95℃,with an optimal primer dosage of 750 pmol,an optimal molecular beacon dosage of 15 pmol,and an optimal reaction time of 30 minutes.The PfAgo fluorescence assay demonstrated high specificity.The LAMP assay identified an optimal amplification temperature of 61 ℃,an optimal reaction time of 40 minutes,an optimal final concentration of dNTP of 1.2 mmol·L-1,an optimal final concentration of manganese ion of 10 mmol·L-1,and an optimal ratio of internal to external primers of 4∶1.Its sensitivity was as low as 101 copies·μL-1,with no cross-reaction with other samples,indicating good specificity.When LAMP was combined with PfAgo fluorescence detection method,the sensitivity achieved at 101 copies·μL-1,and specificity assay results indicated that the method was highly specific.The PPV-LAMP-PfAgo fluorometric method established in this study offers high sensitivity and specificity,with the advantages of sensitivity and rapidity.It provides a new technical approach for the detection of PPV at the clinical grassroots level.
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