- Methodology article
- Open Access
Development of a nanoparticle-assisted PCR (nanoPCR) assay for detection of mink enteritis virus (MEV) and genetic characterization of the NS1 gene in four Chinese MEV strains
© Wang et al.; licensee BioMed Central. 2015
- Received: 9 August 2014
- Accepted: 18 December 2014
- Published: 13 January 2015
Mink enteritis virus (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. Nanoparticle-assisted polymerase chain reaction (nanoPCR) 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 NS1 gene, which encodes nonstructural protein 1. Under optimized conditions, the MEV nanoPCR assay had a detection limit of 8.75 × 101 copies recombinant plasmids per reaction, compared with 8.75 × 103 copies for conventional PCR analysis. Moreover, of 246 clinical mink samples collected from five provinces in North-Eastern China, 50.8% were scored MEV positive by our nanoPCR assay, compared with 32.5% for conventional PCR. Furthermore no cross reactivity was observed for the nanoPCR assay with respect to related viruses, including canine distemper virus (CDV) and Aleutian mink disease parvovirus (AMDV). 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.
- Nanoparticle-assisted PCR
- Mink enteritis virus
- Nonstructural protein 1 gene
- Genetic characterization
- China type
Mink enteritis virus (MEV), a member of the genus Parvovirus within the family Parvoviridae, and a subspecies of the feline parvovirus (FPV), is a single-stranded DNA virus with a genome length of approximately 5,094 nt [1-3]. The MEV genome contains two major open reading frames (ORFs), a 3’ half ORF encoding the nonstructural proteins NS1 and NS2, and a 5’ half ORF encoding the capsid proteins VP1 and VP2.
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 1949  was followed by the isolation and identification of the viral pathogen and development of a vaccine in 1952 . 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 [4,9-16]. 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 (PCR) has been widely used for the detection of MEV and other viruses  through amplification of the highly conserved NS1 and VP2 genes [13,14] and, together with restriction fragment length polymorphism (RFLP), 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 [18-20], porcine [21-23], human B19 [24,25] and human 4  parvoviruses.
Nanoparticle-assisted PCR (nanoPCR)  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
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 6.0 μL of 2× nanobuffer, 0.6 μL each of the upstream and downstream primers (10 μmol/L), 1.0 μL of extracted DNA or standard plasmid, 0.2 μL of Taq DNA polymerase (5 U/μL) 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, 54.9°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
Specificity of the MEV nanoPCR assay
Diagnosis of MEV by nanoPCR assay
Comparison of the sensitivity and specificity of nanoPCR and conventional PCR analysis for detection of MEV in fecal samples
DNA sequencing and phylogenetic analysis
MEV is an important viral pathogen in the mink industry, causing high morbidity and mortality worldwide, and for which there are no effective treatments [32,33]. 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 NS1 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 [14,34], 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 246 field samples in this study, 125 (50.8%) 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 VP2 gene sequences . As shown in the phylogenetic tree, The strains MEV Jlin/2010, MEV-SDNH, MEV SD07/09, and MEV SD12/01 were classified into a samll MEV clade, named China type, in the FPLV/MEV cluster. The nucleotide divergence of the NS1 gene between strains in the China type clade was between 0.1% to 0.5%, and that between China type and other carnivore parvoviruses was between 0.7% to 1.8%, with the exception of the natural recombination virus strain MEV LN-10 strain (data not shown). Specifically, the 357G, 516A, 570 T, 897G, 999A, and 1149G nucleotide residues in the NS1 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.
Viral strains and clinical samples
The viruses (MEV, CDV and AMDV) and 10 experimentally infected samples used in this study have been described in previous reports [4,35]. Animal experiments were approved by the Institute of Special Animal and Plant Sciences of CAAS, and animal experiments were performed in accordance with animal ethics guidelines and approved protocols. Fecal samples were obtained between 2007–2013 in Shandong, Hebei, Liaoning, Heilongjiang, and Jilin provinces, China, from 246 minks showing clinical and pathological signs of enteritis.
Viral DNA/RNA extraction
Fecal samples were collected and stored by our group as previously described . The MEVB or ADMV strain was propagated in the feline kidney F81 cell line in MEM medium. Virus particles were isolated from infected F81 cells when a cytopathic effect was visible about 96 hours after inoculation. Total DNA was extracted from fecal samples and from MEV- or ADMV-infected (positive control) and mock-infected (negative control) cell cultures using a DNA extraction kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. CDV RNA extraction and reverse transcription were performed as previously described .
Primers and construction of recombinant plasmid DNA
NanoPCR and conventional PCR target gene and primers used for amplification of MEV
Primer name a
Genome position b
Melting temperature (°C)
MEV conventional PCR analysis was carried out using a primer set (P1 and P2, see Table 2) yielding a PCR product with a predicted length of 194 bp. PCR was carried out in a 20 μL reaction volume containing 1 μL extracted DNA or standard plasmid, 10 μL of 2× EasyTaq PCR SuperMix containing EasyTaq DNA polymerase, deoxynucleoside triphosphate (dNTP) and buffer (TransGen Biotech Company, Beijing, China), 7 μL ddH2O, and 1 μL of each of primers P1 and P2 (10 μM). The amplification regime was 5 min at 94°C followed by 31 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s, with a final elongation for 5 min at 72°C. PCR was carried out in a Life Express Thermal Cycler (HANGZHOU BIOER TECHNOLOGY CO., LTD, China). PCR products were subjected to electrophoresis on a 2% agarose gel.
Optimization of MEV nanoPCR assay conditions
Optimization of the annealing temperature, plasmid DNA volume and primer volume for the MEV nanoPCR assay was carried out using the same primer pair as in conventional PCR for the MEV nanoPCR assay. Annealing temperatures in the Life Express Thermal Cycler ranged from 48°C to 60°C, the plasmid DNA volumes ranged from 0.1 to 1.8 μL, and the primer volumes ranged from 0.1 to 1.0 μL in increments of 0.1 μL. Products were visualized on 2% agarose gels at a voltage of 250 V for 15 min. The nanoPCR Kit (NPK02) was purchased from GREDBIO (Weihai, China). Gel quantification analysis of all bands was carried out using ImageJ 1.46r software (National Institutes of Health, Bethesda, MA, USA).
Sensitivity of MEV nanoPCR assay
The limits of detection of for the MEV nanoPCR assay detection were compared with conventional PCR using a 10-fold dilution series of the pEASY-T1-MEV-NS1 plasmid (ranging from 8.75 × 108 to 8.75 × 100 copies/μL), and using ddH2O was used as the negative control. PCR products were subjected to electrophoresis on a 2% agarose gel.
Specificity of MEV nanoPCR assay
Cross-reaction of the MEV nanoPCR assay with AMDV DNA and CDV cDNA was evaluated using pEASY-T1-MEV-NS1 as the positive control, and DNA extracted from fecal samples of healthy minks as the negative control. PCR products were subjected to electrophoresis on a 2% agarose gel.
Detection of MEV in clinical samples
The sensitivity of the detection of MEV nanoPCR and conventional PCR assays was compared in clinical fecal samples from 246 minks in five provinces in North-Eastern China during the years 2007–2013. The location sources and the number of samples were as follows: Shandocng (122), Liaoning (31), Jilin (35), Heilongjiang (18), and Hebei (40) provinces. In addition, 10 fecal samples from experimentally infected animals were selected. Four of the positive products from the samples were sequenced.
NS1 gene sequencing and phylogenetic analysis
Nucleotide sequence accession numbers of MEV, CPV and FPLV isolates analyzed in this study
The study was supported by Jilin Provincial Special Economic Animal Biological Products Technology Innovation Center, Jilin Provincial Natural Science Foundation (No. 20140101029JC), and Jilin Provincial Key Science and Technology Project Fund (No. 20140204066NY and No. 20150204021NY).
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