基于MALDI-TOF核酸质谱的11种牛源病毒检测方法的建立  

Development of a MALDI-TOF nucleic acid mass spectrometrybased detection method for eleven viruses

作　　者：帅江冰 张泽林 宋士琦 韩笑 曾若雪 陈吴健 郭悠然 郭惠民 张晓峰 SHUAI Jiang-bing;ZHANG Ze-in;SONG Shi-qi;HAN Xiao;ZENG Ruo-xue;CHEN Wu-jian;GUO You-ran;GUO Hui-min;ZHANG Xiao-feng(Hangzhou Customs Technical Center,Hangzhou 311202,China;Zhejiang Academy of Science and Technology for Inspection and Quarantine,Hangzhou 310016,China;Tonglu County Agriculture and Rural Bureau,Hangzhou 311599,China;Zhejiang Digena Diagnostic Technology Co.,Ltd.,Hangzhou 311100,China)

机构地区：[1]杭州海关技术中心,浙江杭州311202 [2]浙江省检验检疫科学技术研究院,浙江杭州310016 [3]浙江省杭州市桐庐县农业农村局,浙江杭州311599 [4]浙江迪谱诊断技术有限公司,浙江杭州311100

出　　处：《中国预防兽医学报》2025年第1期39-47,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2021YFF0602800);浙江省重点研发计划项目(2021C02060)。

摘　　要：为建立能高效同步鉴定牛腺病毒3型(BAV-3)、牛细小病毒1、2和3型(BPV-1、BPV-2、BPV-3)、牛病毒性腹泻病毒(BVDV)、狂犬病毒(RV)、呼肠孤病毒3型(REO-3)、牛副流感病毒3型(BPIV-3)、蓝舌病毒(BTV)、牛呼吸道合胞体病毒(BRSV)以及牛多瘤病毒(BPyV)等牛源病毒的基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)高通量多目标检测技术,本研究根据上述11种病原基因组的高保守区,分别设计多重PCR引物及对应的单碱基延伸探针,采用方阵法优化反应体系后初步建立牛源病毒的MALDI-TOF核酸质谱检测体系。利用该方法以靶标病毒质粒标准品混合物及非靶标病毒核酸为模板进行特异性试验,利用50拷贝/µL、25拷贝/µL、12.5拷贝/µL、6.3拷贝/µL、3.1拷贝/µL、1.6拷贝/µL靶标病毒核酸质粒标准品进行敏感性试验,利用106拷贝/µL、104拷贝/µL、102拷贝/µL靶标病毒核酸重组质粒标准品于同一时间和不同时间进行了稳定性试验。特异性试验结果显示,该方法仅对11种靶标病毒产生阳性信号峰,而与其他病原体无交叉反应,表明该方法特异性较强。敏感性试验结果通过probit模型进行概率回归检测,结果显示每种病毒最低检测限在8.69拷贝/µL~38.04拷贝/µL(99%概率),表明该方法敏感性较高。重复性试验结果显示,体系中每种靶标在高、中、低质粒浓度时的批内及批间重复性试验结果的符合率均达100%,表明该方法的稳定性较高。利用该MALDI-TOF核酸质谱检测体系对血液类生物制品及临床样品进行检测,同时采用各病毒的荧光定量PCR(qPCR)检测,结果显示130份样品中总计检出58份阳性,其中包括43份BPV-3、33份BPV-2、16份BPyV、6份BVDV阳性,总阳性率分别为44.0%、33.1%、25.4%、12.3%和4.6%,与各病毒qPCR结果的符合率均达100%。本研究基于MALDI-TOF核酸质谱技术首次建立了11种牛病毒的多重检测方法,为牛源病毒的快速检测及�To establish a high-throughput multi-target mass spectrometry(MALDI-TOF MS)system for the simultaneous identification of bovine adenovirus type 3(BAV-3),bovine poliovirus types 1,2 and 3(BPV-1,BPV-2,and BPV-3),bovine viral diarrhea virus(BVDV),rabies virus(RV),reovirus echovirus 3(REO-3),bovine parainfluenza virus 3(BPIV-3),bluetongue virus(BTV),bovine respiratory syncytial virus(BRSV),and bovine polyomavirus(BPyV),and MALDI-TOF MS has been developed.In this study,we designed multiplex PCR primers and corresponding single-base extension probes according to the highly conserved regions of the genomes of the above eleven pathogens and initially established the MALDI-TOF MS of bovine viruses by using the square-array method to optimize the reaction system.The reaction system was optimized using the square array method,and the MALDI-TOF mass spectrometry detection system for bovine viruses was initially established.Specificity tests were performed using target and non-target viral nucleic acids as templates,while sensitivity tests were performed with 50 copies/μL,25 copies/μL,12.5 copies/μL,6.3 copies/μL,3.1 copies/μL,and 1.6 copies/μL plasmid standards.In addition,stability tests were carried out with 106 copies/μL,104 copies/μL,and 102 copies/μL plasmid standards.The results of the specificity test showed that the method produced positive signal peaks only for eleven target viruses,and there was no cross-reactivity with the detection of other pathogens,indicating that the specificity of the method was good.The sensitivity test results were probabilistically regressed by the probit model,which showed that the lowest detection limit of each virus plasmid standard was between 8.69 copies/μL-38.04 copies/μL(99%probability),indicating that the method has high sensitivity.Repeatability test results showed that each target's intra-batch positive compliance rate was 100%at high,medium,and low plasmid concentrations,indicating the method has high stability.This MALDI-TOF nucleic acid mass spectrometry detection s

关 键 词：MALDI-TOF核酸质谱 牛血液制品 病毒 

分 类 号：S852.65[农业科学—基础兽医学]