BACKGROUND
mink enteritis virus , a member of the genus parvovirus within the family parvoviridae, and a subspecies of the feline parvovirus , is a single-stranded dna virus with a genome length of approximately  <dig>  nt  <cit> .
the mev genome contains two major open reading frames , a 3’ half orf encoding the nonstructural proteins ns <dig> and ns <dig>  and a 5’ half orf encoding the capsid proteins vp <dig> and vp <dig>
mev causes mink viral enteritis, an acute and highly contagious disease whose symptoms include violent diarrhea, and which is characterized by high morbidity and mortality  <cit> .
the initial description of the disease in canadian minks in  <dig>  <cit>  was followed by the isolation and identification of the viral pathogen and development of a vaccine in  <dig>  <cit> .
the disease has since been reported in a number of other countries worldwide  <cit> , including china  <cit> , and poses a serious economic threat to the global mink fur farming industry  <cit> .
diagnosis of mev constitutes an important measure for the control of the disease, and although a broad number of approaches have been adopted, they have their own disadvantages  <cit> .
for example, although electron microscopy and virus isolation are highly specific and sensitive, they are often too time-consuming and expensive for routine clinical use.
moreover, the latex agglutination test is rapid but lacks specificity, and the haemagglutination inhibition test requires a continuous supply of fresh erythrocytes and is unsuitable for the detection of non-haemagglutinating mev isolates  <cit> .
conventional polymerase chain reaction  has been widely used for the detection of mev and other viruses  <cit>  through amplification of the highly conserved ns <dig> and vp <dig> genes  <cit>  and, together with restriction fragment length polymorphism , has been used for differentiation of mev vaccine and wild type strains  <cit> .
in addition, real-time pcr have been developed for the detection and quantification of other parvoviruses, including canine  <cit> , porcine  <cit> , human b <dig>  <cit>  and human  <dig>  <cit>  parvoviruses.
nanoparticle-assisted pcr   <cit>  incorporates nanoparticles to improve the specificity and speed of the reaction, and has been successfully applied for the detection of pseudorabies virus  <cit> , bacterial aerosols  <cit> , porcine parvovirus  <cit>  and porcine bocavirus  <cit> .
here we describe the development of a nanopcr-based assay for rapid clinical detection and epidemiological characterization of mev.
RESULTS
optimization of mev nanopcr assay conditions
optimization of the nanopcr assay encompassed adjustment of primer pairs, annealing temperature and the volumes of primer and plasmid dna.
three primer pairs with fragment lengths of 194 bp, 163 bp and 389 bp, respectively, were compared, and based on gel quantification analysis by imagej  <dig> r software, primer pair no.
<dig>  was selected for use in conventional pcr and nanopcr assays .
band density was found to be optimal at an annealing temperature of  <dig> °c, which was chosen for subsequent studies .
using this annealing temperature, band density was found to be maximal at a primer volume of  <dig>  μl   and a plasmid dna volume of  <dig>  μl .
gel quantification analysis of all bands has been carried out using imagej  <dig> r software .figure  <dig>
optimization of annealing temperature , primer concentration , and plasmid dna concentration  for mev nanopcr.
lane m: low dna mass ladder ;  lanes 1–12: the annealing temperatures were 48°c,  <dig> °c,  <dig> °c,  <dig> °c,  <dig> °c,  <dig> °c,  <dig> °c,  <dig> °c,  <dig> °c,  <dig> °c,  <dig> °c, and 60°c, respectively.
lanes 1–10: the primer volumes were  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl, and  <dig>  μl, respectively.
lanes 1–10: the plasmid dna volumes were  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl,  <dig>  μl, and  <dig>  μl, respectively.
based on the results obtained with different annealing temperatures, primer volumes and plasmid dna volumes for the mev nanopcr assay, an optimal 12 μl reaction volume was established, containing  <dig>  μl of 2× nanobuffer,  <dig>  μl each of the upstream and downstream primers ,  <dig>  μl of extracted dna or standard plasmid,  <dig>  μl of taq dna polymerase  and ddh2o up to 12 μl.
the reaction conditions were as follows: 3 min at 94°c, followed by 31 cycles at 94°c for 30 s,  <dig> °c for 30 s and 72°c for 15 s, and a final elongation at 72°c for 10 min.
sensitivity of the mev nanopcr assay
evaluation of the sensitivity of mev nanopcr assay indicated that the detection limit of the mev nanopcr assay  was 100-fold higher than that of conventional pcr analysis .figure  <dig>
evaluation of the sensitivities of nanopcr  and conventional pcr  for the detection of mev
ns1
plasmid dna.
lane m: low dna mass ladder ; lanes 1–9: different mev ns <dig> plasmid dna copies subjected to nanopcr and conventional pcr ; lane 10: blank.
specificity of the mev nanopcr assay
agarose gel electrophoresis analysis indicated no cross reaction of the nanopcr assay with cdv or amdv dnas, nor dna extracted from the tissues of healthy minks, but was positive for mev-infected minks .figure  <dig>
evaluation of the specificity of the mev nanopcr assay.
lane m: low dna mass ladder ; lane 1: mev genome as template; lane 2: cdna of cdv genome as template, lane 3: amdv genome as template, lane 4: dna from fecal samples of healthy mink as template.
diagnosis of mev by nanopcr assay
clinical samples were subjected simultaneously to mev nanopcr and conventional pcr.
eighty samples  were positive for mev by both nanopcr and conventional pcr, and  <dig> samples  were negative by both nanopcr and conventional pcr.
forty five  samples that were positive by nanopcr were negative by conventional pcr, while no sample that was negative by nanopcr was found to be positive by conventional pcr .
compared with the conventional pcr, the relative specificity and sensitivity of nanopcr were  <dig> %  and 100% , respectively.
the ten fecal samples from experimentally infected minks were positive for mev by both nanopcr and conventional pcr.
parts of clinical samples detection by mev nanopcr were shown in figure  <dig> table  <dig>
comparison of the sensitivity and specificity of nanopcr and conventional pcr analysis for detection of mev in fecal samples
nanopcr
conventional pcr
positive
negative
total
percentage of agreement: /246 =  <dig> %; relative sensitivity: 80/80 = 100%; relative specificity: 121/166 =  <dig> %.
detection of mev in clinical samples by nanopcr assay.
lane m: dl <dig> dna maker ; lane 1: mev genome as template; lane 2: plasmid dna as template; lanes 3: negtive control, lanes 4–21: dna from clinical fecal samples as template.
dna sequencing and phylogenetic analysis
sequence analysis indicated high similarity between the products obtained with the nanopcr amplification of the ns <dig> gene of mev  and the reference sequence of mev, indicating that the mev nanopcr is specific.
a phylogenetic tree was constructed by the maximum likelihood method, and the robustness of the phylogenetic analysis was determined by bootstrap analysis with  <dig> replications .
analysis of this tree demonstrated that carnivore parvoviruses were divided into fplv/mev and cpv clusters.
the mev jlin/ <dig>  mev-sdnh, mev sd07/ <dig>  and mev sd12/ <dig> strains were classified into a small mev clade, named the china type, in the fplv/mev cluster.
moreover, mev/ln- <dig>  a natural recombination virus between mink enteritis virus and canine parvovirus  <cit> , was found to be more distant from the small china clade.
in general, strains from the same province shared a common clade.figure  <dig>
phylogenetic analysis of mev with other carnivore parvoviruses based on
ns1
gene nucleotide sequences.
nucleotide sequences were analyzed using the maximum likelihood method and tamura-nei model in mega <dig>  bootstrap values were calculated on  <dig> replicates.
mevs marked by solid triangles were isolated and preserved in our lab.
discussion
mev is an important viral pathogen in the mink industry, causing high morbidity and mortality worldwide, and for which there are no effective treatments  <cit> .
accordingly, to improve epidemiological surveillance and prediction of the severity of mev infection  <cit> , we set out here to develop a simple and rapid diagnostic tool, targeting the conserved mev ns <dig> gene, for the detection and differentiation of mev from other viruses.
a variety of methods currently exist for the detection of mev, including the hemagglutination test and double antibody sandwich elisa for the detection of mev antigen  <cit> , and the haemagglutination inhibition test, serum neutralisation test, and indirect elisa for the detection of mev antibodies  <cit> .
these serological techniques, however, do not distinguish between vaccine or natural infection with wild-type virus as the cause of the antibody response.
moreover, although conventional pcr has been used to identify mev infection  <cit> , it is time-consuming and insensitive, and unsuitable for the detection of low viral loads in clinical samples.
in addition, though lamp assay is simple  <cit> , it is readily subject to contamination.
the present study demonstrated that our nanopcr assay is an effective and time-saving method for detecting mev.
this assay had 100-fold higher analytical sensitivity than conventional pcr, was specific for mev, and exhibited no cross reactivity against other viruses.
of the  <dig> field samples in this study,  <dig>  were positive for mev when assayed by mev nanopcr, indicating the prevalence of mev infection in china.
the results of our phylogenetic analysis, indicating that carnivore parvoviruses were divided into fplv/mev and cpv clusters, is similar to the results of a study based on vp <dig> gene sequences  <cit> .
as shown in the phylogenetic tree, the strains mev jlin/ <dig>  mev-sdnh, mev sd07/ <dig>  and mev sd12/ <dig> were classified into a samll mev clade, named china type, in the fplv/mev cluster.
the nucleotide divergence of the ns <dig> gene between strains in the china type clade was between  <dig> % to  <dig> %, and that between china type and other carnivore parvoviruses was between  <dig> % to  <dig> %, with the exception of the natural recombination virus strain mev ln- <dig> strain  <cit> .
specifically, the 357g, 516a, 570 t, 897g, 999a, and 1149g nucleotide residues in the ns <dig> gene of the china type strains differed from those of all previously described carnivore parvovirus strains.
all new mutations which did not result in amino acid residue replacement were synonymous substitutions.
in summary, we have developed a convenient nanopcr method for the detection of mev that is rapid, sensitive, and specific, and which detects both mev field strains and vaccine strains.
compared with conventional pcr, this nanopcr assay requires minimal laboratory facilities and is relatively simple and inexpensive to perform.
although only limited numbers of clinical samples were used in the present study, further studies will evaluate its performance in different laboratories and with a larger cohort.
CONCLUSIONS
the nanopcr assay developed in this study we have described here will be useful for the detection and epidemiological and pathological characterization of mev.
in addition, our results indicate that the mev china type clade is currently circulating in domestic minks in china.

 mink enteritis virus, a member of the genus parvovirus within the family parvoviridae, and a subspecies of the feline parvovirus, is a single-stranded dna virus with a genome length of approximately  nt. the mev genome contains two major open reading frames, a 3’ half orf encoding the nonstructural proteins ns and ns and a 5’ half orf encoding the capsid proteins vp and vp mev causes mink viral enteritis, an acute and highly contagious disease whose symptoms include violent diarrhea, and which is characterized by high morbidity and mortality. the initial description of the disease in canadian minks in was followed by the isolation and identification of the viral pathogen and development of a vaccine in. the disease has since been reported in a number of other countries worldwide, including china, and poses a serious economic threat to the global mink fur farming industry. diagnosis of mev constitutes an important measure for the control of the disease, and although a broad number of approaches have been adopted, they have their own disadvantages. for example, although electron microscopy and virus isolation are highly specific and sensitive, they are often too time-consuming and expensive for routine clinical use. moreover, the latex agglutination test is rapid but lacks specificity, and the haemagglutination inhibition test requires a continuous supply of fresh erythrocytes and is unsuitable for the detection of non-haemagglutinating mev isolates. conventional polymerase chain reaction has been widely used for the detection of mev and other viruses through amplification of the highly conserved ns and vp genes and, together with restriction fragment length polymorphism, has been used for differentiation of mev vaccine and wild type strains. in addition, real-time pcr have been developed for the detection and quantification of other parvoviruses, including canine, porcine, human b and human parvoviruses. nanoparticle-assisted pcr incorporates nanoparticles to improve the specificity and speed of the reaction, and has been successfully applied for the detection of pseudorabies virus, bacterial aerosols, porcine parvovirus and porcine bocavirus. here we describe the development of a nanopcr-based assay for rapid clinical detection and epidemiological characterization of mev.  optimization of mev nanopcr assay conditions optimization of the nanopcr assay encompassed adjustment of primer pairs, annealing temperature and the volumes of primer and plasmid dna. three primer pairs with fragment lengths of 194 bp, 163 bp and 389 bp, respectively, were compared, and based on gel quantification analysis by imagej r software, primer pair no. was selected for use in conventional pcr and nanopcr assays. band density was found to be optimal at an annealing temperature of °c, which was chosen for subsequent studies. using this annealing temperature, band density was found to be maximal at a primer volume of  μl and a plasmid dna volume of  μl. gel quantification analysis of all bands has been carried out using imagej r software.figure optimization of annealing temperature, primer concentration, and plasmid dna concentration for mev nanopcr. lane m: low dna mass ladder; lanes 1–12: the annealing temperatures were 48°c, °c, °c, °c, °c, °c, °c, °c, °c, °c, °c, and 60°c, respectively. lanes 1–10: the primer volumes were  μl,  μl,  μl,  μl,  μl,  μl,  μl,  μl,  μl, and  μl, respectively. lanes 1–10: the plasmid dna volumes were  μl,  μl,  μl,  μl,  μl,  μl,  μl,  μl,  μl, and  μl, respectively. based on the results obtained with different annealing temperatures, primer volumes and plasmid dna volumes for the mev nanopcr assay, an optimal 12 μl reaction volume was established, containing  μl of 2× nanobuffer,  μl each of the upstream and downstream primers,  μl of extracted dna or standard plasmid,  μl of taq dna polymerase and ddh2o up to 12 μl. the reaction conditions were as follows: 3 min at 94°c, followed by 31 cycles at 94°c for 30 s, °c for 30 s and 72°c for 15 s, and a final elongation at 72°c for 10 min. sensitivity of the mev nanopcr assay evaluation of the sensitivity of mev nanopcr assay indicated that the detection limit of the mev nanopcr assay was 100-fold higher than that of conventional pcr analysis.figure evaluation of the sensitivities of nanopcr and conventional pcr for the detection of mev ns1 plasmid dna. lane m: low dna mass ladder; lanes 1–9: different mev ns plasmid dna copies subjected to nanopcr and conventional pcr; lane 10: blank. specificity of the mev nanopcr assay agarose gel electrophoresis analysis indicated no cross reaction of the nanopcr assay with cdv or amdv dnas, nor dna extracted from the tissues of healthy minks, but was positive for mev-infected minks.figure evaluation of the specificity of the mev nanopcr assay. lane m: low dna mass ladder; lane 1: mev genome as template; lane 2: cdna of cdv genome as template, lane 3: amdv genome as template, lane 4: dna from fecal samples of healthy mink as template. diagnosis of mev by nanopcr assay clinical samples were subjected simultaneously to mev nanopcr and conventional pcr. eighty samples were positive for mev by both nanopcr and conventional pcr, and samples were negative by both nanopcr and conventional pcr. forty five samples that were positive by nanopcr were negative by conventional pcr, while no sample that was negative by nanopcr was found to be positive by conventional pcr. compared with the conventional pcr, the relative specificity and sensitivity of nanopcr were % and 100%, respectively. the ten fecal samples from experimentally infected minks were positive for mev by both nanopcr and conventional pcr. parts of clinical samples detection by mev nanopcr were shown in figure  table comparison of the sensitivity and specificity of nanopcr and conventional pcr analysis for detection of mev in fecal samples nanopcr conventional pcr positive negative total percentage of agreement: /246 =  %; relative sensitivity: 80/80 = 100%; relative specificity: 121/166 =  %. detection of mev in clinical samples by nanopcr assay. lane m: dl dna maker; lane 1: mev genome as template; lane 2: plasmid dna as template; lanes 3: negtive control, lanes 4–21: dna from clinical fecal samples as template. dna sequencing and phylogenetic analysis sequence analysis indicated high similarity between the products obtained with the nanopcr amplification of the ns gene of mev and the reference sequence of mev, indicating that the mev nanopcr is specific. a phylogenetic tree was constructed by the maximum likelihood method, and the robustness of the phylogenetic analysis was determined by bootstrap analysis with replications. analysis of this tree demonstrated that carnivore parvoviruses were divided into fplv/mev and cpv clusters. the mev jlin/ mev-sdnh, mev sd07/ and mev sd12/ strains were classified into a small mev clade, named the china type, in the fplv/mev cluster. moreover, mev/ln- a natural recombination virus between mink enteritis virus and canine parvovirus, was found to be more distant from the small china clade. in general, strains from the same province shared a common clade.figure phylogenetic analysis of mev with other carnivore parvoviruses based on ns1 gene nucleotide sequences. nucleotide sequences were analyzed using the maximum likelihood method and tamura-nei model in mega bootstrap values were calculated on replicates. mevs marked by solid triangles were isolated and preserved in our lab. discussion mev is an important viral pathogen in the mink industry, causing high morbidity and mortality worldwide, and for which there are no effective treatments. accordingly, to improve epidemiological surveillance and prediction of the severity of mev infection, we set out here to develop a simple and rapid diagnostic tool, targeting the conserved mev ns gene, for the detection and differentiation of mev from other viruses. a variety of methods currently exist for the detection of mev, including the hemagglutination test and double antibody sandwich elisa for the detection of mev antigen, and the haemagglutination inhibition test, serum neutralisation test, and indirect elisa for the detection of mev antibodies. these serological techniques, however, do not distinguish between vaccine or natural infection with wild-type virus as the cause of the antibody response. moreover, although conventional pcr has been used to identify mev infection, it is time-consuming and insensitive, and unsuitable for the detection of low viral loads in clinical samples. in addition, though lamp assay is simple, it is readily subject to contamination. the present study demonstrated that our nanopcr assay is an effective and time-saving method for detecting mev. this assay had 100-fold higher analytical sensitivity than conventional pcr, was specific for mev, and exhibited no cross reactivity against other viruses. of the field samples in this study, were positive for mev when assayed by mev nanopcr, indicating the prevalence of mev infection in china. the results of our phylogenetic analysis, indicating that carnivore parvoviruses were divided into fplv/mev and cpv clusters, is similar to the results of a study based on vp gene sequences. as shown in the phylogenetic tree, the strains mev jlin/ mev-sdnh, mev sd07/ and mev sd12/ were classified into a samll mev clade, named china type, in the fplv/mev cluster. the nucleotide divergence of the ns gene between strains in the china type clade was between % to %, and that between china type and other carnivore parvoviruses was between % to %, with the exception of the natural recombination virus strain mev ln- strain. specifically, the 357g, 516a, 570 t, 897g, 999a, and 1149g nucleotide residues in the ns gene of the china type strains differed from those of all previously described carnivore parvovirus strains. all new mutations which did not result in amino acid residue replacement were synonymous substitutions. in summary, we have developed a convenient nanopcr method for the detection of mev that is rapid, sensitive, and specific, and which detects both mev field strains and vaccine strains. compared with conventional pcr, this nanopcr assay requires minimal laboratory facilities and is relatively simple and inexpensive to perform. although only limited numbers of clinical samples were used in the present study, further studies will evaluate its performance in different laboratories and with a larger cohort.  the nanopcr assay developed in this study we have described here will be useful for the detection and epidemiological and pathological characterization of mev. in addition, our results indicate that the mev china type clade is currently circulating in domestic minks in china. 
here we describe a novel nanopcr assay for the clinical detection and epidemiological characterization of mev. this assay is based upon primers specific for the conserved region of the mev ns gene, which encodes nonstructural protein under optimized conditions, the mev nanopcr assay had a detection limit of  ×  copies recombinant plasmids per reaction, compared with  ×  copies for conventional pcr analysis. moreover, of clinical mink samples collected from five provinces in north-eastern china, % were scored mev positive by our nanopcr assay, compared with % for conventional pcr. furthermore no cross reactivity was observed for the nanopcr assay with respect to related viruses, including canine distemper virus and aleutian mink disease parvovirus. phylogenetic analysis of four chinese wild type mev isolates using the nanopcr assay indicated that they belonged to a small mev clade, named “china type”, in the mev/fplv cluster, and were closely clustered in the same location. we anticipate that the nanopcr assay we have described here will be useful for the detection and epidemiological and pathological characterization of mev. mink enteritis virus causes mink viral enteritis, an acute and highly contagious disease whose symptoms include violent diarrhea, and which is characterized by high morbidity and mortality. nanoparticle-assisted pcrmink enteritis virusnonstructural protein genegenetic characterizationchina typeissue-copyright-statement© the author 2015 our results indicate that the mev china type clade is currently circulating in domestic minks in china.
 mink enteritis virus causes mink viral enteritis, an acute and highly contagious disease whose symptoms include violent diarrhea, and which is characterized by high morbidity and mortality. nanoparticle-assisted polymerase chain reaction is a recently developed technique for the rapid detection of bacterial and viral dna. here we describe a novel nanopcr assay for the clinical detection and epidemiological characterization of mev.  this assay is based upon primers specific for the conserved region of the mev ns gene, which encodes nonstructural protein under optimized conditions, the mev nanopcr assay had a detection limit of  ×  copies recombinant plasmids per reaction, compared with  ×  copies for conventional pcr analysis. moreover, of clinical mink samples collected from five provinces in north-eastern china, % were scored mev positive by our nanopcr assay, compared with % for conventional pcr. furthermore no cross reactivity was observed for the nanopcr assay with respect to related viruses, including canine distemper virus and aleutian mink disease parvovirus. phylogenetic analysis of four chinese wild type mev isolates using the nanopcr assay indicated that they belonged to a small mev clade, named “china type”, in the mev/fplv cluster, and were closely clustered in the same location.  our results indicate that the mev china type clade is currently circulating in domestic minks in china. we anticipate that the nanopcr assay we have described here will be useful for the detection and epidemiological and pathological characterization of mev. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords nanoparticle-assisted pcrmink enteritis virusnonstructural protein genegenetic characterizationchina typeissue-copyright-statement© the author 2015 