The Development And Application Of A Duplex PCR Method For The Detection Of Aleutian Mink Disease

Aleutian Mink Disease(AMD),one of the three epidemic diseases of fur animals(canine distemper fever,viral enteritis,and Aleutian disease),which caused huge economic losses to the aquaculture industry.Up to now,there is no effective drug or vaccine to prevent and cure the disease for the reason of its special pahtogenesis.The only preventive method of AD was to purify and eliminate the positive mink.At present,the Counter Immunoelectrophoresis assay(CIEP)for antibody detection is mainly used in the detection of the disease at the early onset of Aleutian's disease,and it is still positive for the detection of some rehabilitation leech,which is not conducive to the breeding of resistance to Aleutian disease.And in recent years,with the continuous transmission and variation of Aleutian disease,common pathogen detection methods often appear missed.The advantages of duplex PCR method include time-saving,labor-saving,simple,rapid,sensitivety and specificity in detecting multiple pathogenic microorganism infections.It has been widely used in the identification and detection of various viruses.AMDV has been widely spread among mink flocks in China.However,the sensitive and specific duplex PCR method has not been established.In this study,after analysis of the published AMDV sequences in GenBank,two pairs of specific primers including NS1-F/NS1-R(located in the conserved region of NS1 gene)and VP2-F/ VP2-R(located in the conserved region of VP2 gene)respectively were designed to establish a duplex PCR assay.The target gene of NS1 and VP2 could be amplified by single PCR method.The NS1 target fragment was 568 bp and the VP2 target fragment was 412 bp.Then we recovered the target gene and ligated to PMD18-T vector and transformed to Dh5?cells.After the identification and sequencing of the recombinant plalmid,it was proved that the amplified fragments amplified belonged to AMDV.The specificity and sensitivity of primers were tested by single PCR method first.The results showed that the two pairs of primers were specific and sensitive.The PCR method can detect at lowest 10 pg/?L AMDV DNA.On the basis of the single PCR reaction conditions,we optimized the reaction conditions of duplex PCR method.Besides,the amplification fragment of NS1 gene was 568 bp and the amplified fragment of VP2 gene was 412 bp.The two target bands can be devided clearly by visual judgement after running on 1% agarose gel electrophoresis.The duplex method was used to detect samples from one standard flocks in different times.In September 2016,964 samples were collected and 54 samples were positive,and the total positive rate was 5.6%.In December 2016,6996 samples were collected and 218 samples were positive,and the total positive rate was 3.12%.In June 2017,2647 samples were collected and 77 samples were positive,and the total positive rate was 2.91%.Out of3356 samples collected in November 2017,85 were detected as positive,and the total positive rate was 2.53%.According to the detection outcome of the four test for the standard flock,and combining with the actual condition of the flock,with the corresponding biosecurity measures.Finally,the positive rate of AMDV in the standard mink flock decreased significantly.