To establish a microdroplet digital PCR(ddPCR)detection method for the aleutian mink disease virus(AMDV),this study designed specific primers and probes based on the conserved region of the AMDV VP2 gene.The AMDV VP2 gene was amplified by PCR and cloned into the pMD19-T vector.The recombinant plasmid pMD19-T-AMDV,identified by PCR and sequencing,was the plasmid standard used as a template.A preliminary ddPCR method for detecting AMDV was established by optimizing various reaction conditions.AMDV,mink enteritis parvovirus,canine distemper virus,canine parvovirus,and mink influenza virus were used to evaluate the specificity of the established ddPCR method.Using 1.43×107 copies/μL-1.43×100 copies/μL of the recombinant plasmid as a template,the sensitivity of this method was evaluated using optimized ddPCR detection and fluorescence quantitative PCR(qPCR)detection in literature.Inter-and intra-batch repeatability experiments were conducted.The coefficient of variation(CV value)was used to evaluate the effect of repeatability on this method.The optimization results showed that this method's optimal primer final concentration is 400nmol/L(1.2μL).The final concentration of the probe is 200nmol/L(0.6μL),and the annealing temperature is 60℃.The specificity test showed that this method can detect AMDV and the plasmid standard,while other related viruses showed negative results.The sensitivity test showed that the detection limit of this method for the plasmid standard is 1.43 copies/μL.The detection limit of the qPCR for plasmid standard is 1.43×102 copies/μL.The former has 100 times higher sensitivity than the latter.The variation coefficient for intra-and inter-batch repeatability tests was less than 3%.Seventy samples of liver,spleen,kidney,environmental swab,and anal swab collected from diseased minks were subjected to ddPCR and qPCR detection.The results showed that the detection rate of AMDV by ddPCR was 28.33%(17/70).In contrast,the detection rate of qPCR is 15.7%(11/70).The positive agreement rate is 64.7%,the negative agreement rate is 89.83%,and the total agreement rate is 91.42%.The results showed that the ddPCR method established in this study has strong specificity,high sensitivity,and good repeatability.The ddPCR can detect AMDV in various clinical samples and samples with extremely low viral load.This study provides a robust technical means for detecting early AMDV infection,epidemiological investigation,prevention,and control of AMD.
droplet digital PCRdetectionaleutian mink disease virus
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