猪圆环病毒2型数字微滴PCR检测技术的建立及应用  

Development of droplet digital polymerase chain reaction assay for Porcine circovirus type 2

作　　者：吴朦晨 金晨晨 何永强 曾若雪 陈小金 程昌勇[4] 帅江冰 董志珍 宋厚辉[4] 俞晓平[1] 张晓峰 WU Meng-chen;JIN Chen-chen;HE Yong-qiang;ZENG Ruo-xue;CHEN Xiao-jin;CHENG Chang-yong;SHUAI Jiang-bing;DONG Zhi-zhen;SONG Hou-hui;YU Xiao-ping;ZHANG Xiao-feng(College of Life Sciences,China Jiliang University,Hangzhou 310018,China;Zhejiang Academy of Science and Technology for Inspection and Quarantine,Hangzhou 310016,China;Animal and Plant and Foodstuffs Inspection Center,Tianjin Customs,Tianjin 300461,China;College of Animal Science and Technology,Zhejiang A&F University,Hangzhou 311300,China)

机构地区：[1]中国计量大学生命科学院,浙江杭州310018 [2]浙江省检验检疫科学技术研究院,浙江杭州310016 [3]天津海关动植物与食品检测中心,天津300461 [4]浙江农林大学,浙江杭州311300

出　　处：《中国预防兽医学报》2021年第10期1057-1062,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省重点研发计划项目(2021C02060、2015C02044)。

摘　　要：为建立猪圆环病毒2型(PCV2)的微滴式数字聚合酶链式反应(ddPCR)检测方法,本研究针对PCV2ORF1基因设计引物和探针,经各反应条件的优化,确定反应体系中引物和探针终浓度分别为0.7μmol/L和0.25μmol/L,且退火温度为59℃时,ddPCR方法具有最佳的扩增效率(96%)。特异性试验结果显示,该ddPCR方法除了对PCV2检测结果为阳性外,对PCV1、PCV3和其它常见猪病病原的检测均未见阳性微滴,特异性较强。以10倍倍比稀释的重组质粒标准品pUC57-NA-ORF1为模板,利用建立的PCV2 ddPCR检测技术进行敏感性试验,结果显示,ddPCR的最低检测限达2.93拷贝/μL,比荧光定量PCR(qPCR)高10倍以上,敏感性较高。批内和批间重复性试验结果显示,其变异系数均小于10%,重复性较好。利用建立的PCV2 ddPCR对24份猪组织样品和70份宠物饲料样品进行检测,结果显示,该ddPCR方法对猪淋巴结、脾、肺和肾脏等组织样品的检测结果与qPCR的检测结果一致,均检出10份阳性样品。但ddPCR对饲料样品的检测更为灵敏,共检出PCV2阳性样品14份,其中有5份样品的qPCR检测结果为阴性,表明本研究建立的ddPCR方法敏感性、抗干扰性和可靠性更高。本研究首次建立了PCV2 ddPCR检测方法,为饲料等复杂基质样品中PCV2的检测提供了有力技术手段。In this study,a detection assay based on droplet digital polymerase chain reaction(ddPCR)system was developed for monitoring the infection of porcine circovirus type 2(PCV2)by targeting its ORF1 gene.The reaction conditions were optimized,and the final concentrations of primer and probe were 0.7μmol/L and 0.25μmol/L,respectively,and the optimized annealing temperature was 59℃.Positive signals were obtained only for PCV2 genome,but not for PCV1,PCV3 and other swine pathogens by this ddPCR system,suggesting its specificity.The lower limit of detection of this ddPCR assay was estimated to be 2.93 copies/μL,which was 10 times more sensitive than a conventiaonal quantitative PCR(q PCR)method.The coefficient of variation of the intra-and inter-assay repeatability test of this method was less than 10%,which suggests this method is reproducible.A panel of field samples including 24 tissue samples and 70 pet feed samples were analyzed by the dd PCR and q PCR,respectively.The results of tissue samples detected by dd PCR assay were consistent with those of q PCR,and both methods detected10 positive samples.However,fourteen pet feed samples for PCV2 were detected positive by the dd PCR assay,of which 5 samples were detected negative by q PCR,indicating that the dd PCR assay was a much more sensitive,anti-interference,reliable and powerful method for PCV2 detection in complex matrix samples such as feed.

关 键 词：数字微滴PCR 猪圆环病毒2型 最低检测限 

分 类 号：S852.65[农业科学—基础兽医学]