Establishment And Application Of Real-time Quantitative PCR And IELISA For Mink Circovirus

Mink circovirus(MiCV)is a novel circovirus associated with mink diarrhea,which was discovered in 2014.At present,there are few studies on the etiology,prevalence and pathogenesis of MiCV.The research on virus transmission has not been reported,and the economic impact of MiCV on mink farming is still unknown.Therefore,the establishment of efficient,accurate and specific detection methods for MiCV is particularly important.However,only PCR and RPA method were currently reported for MiCV detection.In order to quantitatively detect MiCV in the infected minks,a pair of specific primer based on Cap gene were designed,and used to construct a standard plasmid to establish the real time quantitative PCR(RT-qPCR).All parameters were following:the standard curve equation is Y=-3.155X+39.202,the amplification efficiency is 107.447%,the slope is-3.155,and correlation coefficient(R~2)is 0.974.The results proved that the method had good specificity,high sensitivity,and good repeatability,and the minimum detectable 10~1 copies/?L.The RT-qPCR method was used to detect samples from different provinces,including faeces and serum samples.The positive rates were 52.88%in Shandong,67.90%in Hebei,58.46%in Liaoning,38.46%in Jilin,30.30%in Heilongjiang,and the total positive rate was 49.38%.The results showed that the infection rate of MiCV in China was high,especially in Hebei province.At the same time,through the quantitative detection method,virus load of infected minks in different organs,as following lung,liver,heart,spleen,etc.It was found that MiCV was detected in the tissues and organs of mink artificial infection and natural infection,and it was considered that the heart may be the preferred tissue material for MiCV detection.Secondly,6 complete genome sequences from different provinces were obtained from positive templates,and the whole genome sequence and analysis were amplified.The nucleotide and deduced amino acid homology and phylogenetic tree analysis of ORF1 and ORF2 of MiCV showed that the sequence length was 1753 bp,MiCV strain ORF1 nucleotide and amino acid homology respectively in 99.3%-99.9%and 99.3%-100%and between ORF2;nucleotide and amino acid sequence homology between 98.2%-100%and 98.7%-100%98.2%,respectively.The results showed that the Cap gene was well conserved and the rate of variation was not very high in all the MiCV strains found at present.After that,to evaluate the Sero epidemiological characteristics,a indirect ELISA detection method of MiCV was established by constructing prokaryotic expression recombinant protein coated antigen.Through checkerboard titration and optimization of conditions,we determined the concentration of antigen coated 5?g/mL,the serum dilution 1:200 and A/G protein dilution1:10000.Comparing the indirect ELISA method with the Western Blot method,the sensitivity and specificity were 92.31%and 91.67%respectively,and the coincidence of the two detection methods was 0.838.It is proved that the method has the good repeatability.Then the new indirect ELISA method was used to detect the serum samples from five provinces in Heilongjiang,Jilin,Liaoning,Shandong and Hebei.The positive rates were as follows:26.79%in Shandong,33.81%in Hebei,25%in Liaoning,27.87%in Jilin and 13.04%in Heilongjiang,and the total positive rate was 23.87%.Among 24 farms,21 farmss was detected in the presence of MiCV antibody,the test results show that the MiCV has a wide range of infection in the main aquaculture areas of mink in China.This study examined the infection of MiCV in two main mink breeding provinces in China from the following aspects:virus DNA and serum antibody.To provide basic information for further development of epidemiology and prevention and control of MiCV pathogenesis.