犬细小病毒高效纳米PCR检测方法的建立及初步应用
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摘要
为建立灵敏、快速的犬细小病毒(CPV)检测方法,本研究针对CPV VP2基因保守序列设计一对能扩增574 bp片段的特异性引物,对纳米PCR的退火温度、引物浓度等反应条件进行了优化,并对其特异性和灵敏度进行了评估。结果表明,所建立的纳米PCR特异性和灵敏性良好,最低核酸检出量为2拷贝·μL~(-1),其敏感性比常规PCR高1 000倍。分别采用纳米PCR和常规PCR,对广西、北京、吉林送检的75份疑似CPV的临床样品进行检测,CPV阳性率分别为89.3%(67/75)和70.7%(53/75),表明该方法具有更高的敏感性,适用于CPV低含量临床样品的检测。本方法的建立为CPV感染的早期快速、灵敏、准确的诊断提供了新方法,为CPV的防控奠定基础,具有重大的临床应用意义和价值。
In order to establish a rapid and sensitive nanoparticle-assisted PCR(nano PCR) assay for detection of canine parvovirus(CPV), a pair of primers was designed based on the conserved region of the VP2 gene to amplify 574 bp fragment. The annealing temperature, primer concentration and other reaction conditions of nano PCR were optimized, and its specificity and sensitivity were evaluated. The results showed that the established nano PCR had good specificity and sensitivity, with a minimum nucleic acid detection amount of 2 copies·μL~(-1), and its sensitivity was 1 000 times higher than that of conventional PCR. Seventy-five clinical samples collected from Beijing, Guangxi and Jilin were tested, the positive rate of CPV was 89.3%(67/75) and 70.7%(53/75) by nano PCR and conventional PCR, respectively. The results showed that the method had higher sensitivity and was suitable for the detection of low amount of CPV in clinical samples. The established nano PCR in this study provided a new method for rapid, sensitive and accurate diagnosis of CPV infection in the early stage,which laid a foundation for the prevention and control of CPV and had significant value in clinical application.
In order to establish a rapid and sensitive nanoparticle-assisted PCR(nano PCR) assay for detection of canine parvovirus(CPV), a pair of primers was designed based on the conserved region of the VP2 gene to amplify 574 bp fragment. The annealing temperature, primer concentration and other reaction conditions of nano PCR were optimized, and its specificity and sensitivity were evaluated. The results showed that the established nano PCR had good specificity and sensitivity, with a minimum nucleic acid detection amount of 2 copies·μL~(-1), and its sensitivity was 1 000 times higher than that of conventional PCR. Seventy-five clinical samples collected from Beijing, Guangxi and Jilin were tested, the positive rate of CPV was 89.3%(67/75) and 70.7%(53/75) by nano PCR and conventional PCR, respectively. The results showed that the method had higher sensitivity and was suitable for the detection of low amount of CPV in clinical samples. The established nano PCR in this study provided a new method for rapid, sensitive and accurate diagnosis of CPV infection in the early stage,which laid a foundation for the prevention and control of CPV and had significant value in clinical application.
引文
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[3] PéREZ R,BIANCHI P,CALLEROS L,et al.Recent spreading of a divergent canine parvovirus type 2a (CPV-2a) strain in a CPV-2c homogenous population[J].Vet Microbiol,2012,155(2-4):214-219.
[4] BUONAVOGLIA C,MARTELLA V,PRATELLI A,et al.Evidence for evolution of canine parvovirus type 2 in Italy[J].J Gen Virol,2001,82(12):3021-3025.
[5] DECARO N,BUONAVOGLIA C.Canine parvovirus—A review of epidemiological and diagnostic aspects,with emphasis on type 2c[J].Vet Microbiol,2012,155(1):1-12.
[6] 张仁舟,杨松涛,冯昊,等.中国国内首次检测到犬细小病毒CPV-2c[J].中国病原生物学杂志,2010,5(4):246-249,275.ZHANG R Z,YANG S T,FENG H,et al.The first detection of canine parvovirus type 2c in China[J].Journal of Pathogen Biology,2010,5(4):246-249,275.(in Chinese)
[7] PALERMO L M,HAFENSTEIN S L,PARRISH C R.Purified feline and canine transferrin receptors reveal complex interactions with the capsids of canine and feline parvoviruses that correspond to their host ranges[J].J Virol,2006,80(17):8482-8492.
[8] LI H K,HUANG J H,LV J H,et al.Nanoparticle PCR:nanogold-assisted PCR with enhanced specificity[J].Angew Chem Int Ed Engl,2005,44(32):5100-5103.
[9] SHEN C C,YANG W J,JI Q L,et al.NanoPCR observation:different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions[J].Nanotechnology,2009,20(45):455103.
[10] LI H X,ROTHBERG L.Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles[J].Proc Natl Acad Sci U S A,2004,101(39):14036-14039.
[11] MA X J,CUI Y C,QIU Z,et al.A nanoparticle-assisted PCR assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and gene-deleted vaccine strains[J].J Virol Methods,2013,193(2):374-378.
[12] DECARO N,DESARIO C,BEALL M J,et al.Detection of canine parvovirus type 2c by a commercially available in-house rapid test[J].Vet J,2010,184(3):373-375.
[13] HIRASAWA T,KANESHIGE T,MIKAZUKI K.Sensitive detection of canine parvovirus DNA by the nested polymerase chain reaction[J].Vet Microbiol,1994,41(1-2):135-145.
[14] DECARO N,ELIA G,MARTELLA V,et al.A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 in the feces of dogs[J].Vet Microbiol,2005,105(1):19-28.
[15] WANG J K,CHENG Y N,ZHANG M,et al.Development of a nanoparticle-assisted PCR (nano PCR) assay for detection of mink enteritis virus (MEV) and genetic characterization of the NS1 gene in four Chinese MEV strains[J].BMC Vet Res,2015,11:1.
[16] WANG X L,BAI A Q,ZHANG J,et al.A new nano PCR molecular assay for detection of porcine bocavirus[J].J Virol Methods,2014,202:106-111.
[17] CUI Y,WANG Z,MA X,et al.A sensitive and specific nanoparticle-assisted PCR assay for rapid detection of porcine parvovirus[J].Lett Appl Microbiol,2014,58(2):163-167.
[18] GABRIEL S,RASHEED A K,SIDDIQUI R,et al.Development of nanoparticle-assisted PCR assay in the rapid detection of brain-eating amoebae[J].Parasitol Res,2018,117(6):1801-1811.