基于毛细管电泳的7种猪源性疫病的多重PCR检测方法的建立

Establishment and application of multiplex PCR-capillary electrophoresis for 7 swine diseases

为建立一种基于毛细管电泳技术,可同时高效地检测7种常见猪源性疫病(猪链球菌、猪瘟病毒、日本脑炎病毒、猪繁殖与呼吸综合征病毒、副猪嗜血杆菌、猪轮状病毒、沙门氏菌)的高通量快速检测方法,本研究将多重PCR与毛细管电泳相结合,针对各毒株基因保守序列分别设计7对特异性引物,在每条引物的5′端加上通用引物标签序列,构成特异性嵌合引物。进行梯度PCR扩增以优化退火温度,使用正交试验以进行特异性嵌合引物浓度的优化。取107~10-1 copies·μL-1浓度梯度的标准品进行检测,以评价该方法的灵敏性。选取7种病原的DNA或cDNA,进行随机分组作为模板进行扩增,检测交叉反应性。以猪流行性腹泻病毒、猪传染性胃肠炎病毒、构建的非洲猪瘟质粒、大肠埃希菌等其他重要病原的DNA或cDNA进行扩增,评价该方法的特异性。使用梯度稀释的7重混合样品进行扩增,每个稀释度进行3次扩增,以评价该方法的重复性。结果显示:该方法的最优退火温度为58.3℃,对7种病原进行同时检出的检测限度为103 copies·μL-1,各种对照样品均出现了特异性峰值,并且所有的阴性对照样品均未扩增,也未出现交叉反应性,重复试验之间数据差异变化微小,63份临床样品检测结果与国标或行业检测标准的检测结果完全一致。以上实验结果显示,所建立的方法具有高通量、特异性好、灵敏度高、方便快速等优点,可用于临床鉴别诊断和流行病学调查,有较高的临床应用价值。

The aim of this study was to develop a multiple PCR assay on a basis of capillary electrophoresis for simultaneous detection of seven pathogens of swine infectious diseases,including Streptococcus suis(SS),Classical swine fever virus(CSFV),Japanese encephalitis virus(JEV),Porcine reproductive and respiratory syndrome virus(PRRSV),Haemophilus parasuis(HPS),Porcine rotavirus(PoRV),Salmonella(SE).Firstly the conservative sequences of the 7 reference pathogens were analyzed to design 7 pairs of specific primers.Subsequently,the specific chimeric primers were obtained by adding universal primer sequences in 5′end of each specific primer.Gradient PCR amplification was adopted to optimize annealing temperature.The primer concentrations were optimized by orthogonal test.The sensitivity test was performed by using serially diluted standards(107-10-1 copies·μL-1).Cross reactivity test was performed by using random mixed templates that orginate from cDNA or DNA of these 7 pathogens.Specificity test was performed by detection of some other common pathogens,including PEDV,TGEV,ASFV plasmid,E.coli.Repeatability test was performed by using serially diluted standards as templates to be amplified triplicates.The results indicated that the optimum annealing temperature was 58.3℃.Detection limit of this assay was 103 copies·μL-1,when 7 positive results were visible from identical lane.With a variety of control samples,the results of positive samples showed specificity peak in contrast of the negative results of all the negative control samples.In addition,neither cross reactivity nor unspecific amplification were detected within these 7 kinds of random mixed templates.the differences among triplicate experiments were small and low.The test results of the 63 clinical samples were exactly the same with the results obtained by national standards.As is indicated in the results,it is an effective toll applied to differential diagnosis rapidly for clinical samples and epidemiological investigation.