摘要
本研究利用叠氮溴化丙锭(PMA)与多重荧光定量PCR(qPCR)技术相结合,建立了一种可以实现非洲猪瘟病毒(African swine fever virus,ASFV)感染性与非感染性(活/死病毒)鉴别的检测方法。通过对ASFV p72、CD2v、MGF110-14L基因保守序列设计特异性引物及探针,建立p72、CD2v、MGF110-14L基因的多重qPCR检测方法。结果显示,本研究建立的多重qPCR检测方法具有较强特异性,与其他常见猪病毒性原无交叉反应;同时,该方法具有较高灵敏度,p72、CD2v、MGF110-14L基因的重组质粒标准品最低检出限均为102copies/μL。为弥补qPCR技术不能有效区分样品中活/死病毒的缺陷,本研究将构建的多重qPCR与PMA技术相结合。当加入PMA终浓度为10μg/mL、暗孵育10 min、光照强度40 W、光照15 min时,PMA能够抑制灭活病毒基因的扩增且对活病毒基因的扩增无影响;对比普通qPCR与多重PMA-qPCR的灵敏度可知,两者的灵敏度均为102copies/μL;将多重PMA-qPCR方法用于23份实际样品检测,结果显示,检测活病毒的准确率为100%,说明该方法能有效区分临床样品中具有感染性的ASFV。
In this study,the combination of the PMA and qPCR technologies were used to establish a method for detection and identification of infectious and non-infectious(live or dead)African swine fever virus(ASFV).By designing specific primers and probes for the conserved sequences of the ASFV p72,CD2v and MGF110-14L genes,a multiplex qPCR method for detection of P72,CD2v and MGF110-14L genes were established.The results showed that the specificity of our multiplex qPCR was high and it had no cross-reactivity with other swine diseases.Its sensitivity was also high,and its minimum detection limit of recombinant plasmid standards for the p72,CD2v,and MGF110-14L genes was 102 copies/μL.To compensate for the inability of the qPCR technology to effectively distinguish between live/dead virus defects in samples,this study combined the optimally processed PMA with qPCR analysis.The results showed that,when PMA was added at a final mass concentration of 10μg/mL,dark incubation for 10 min,light intensity of 40 W,and light for 15 min,PMA could inhibit the expansion of the dead virus gene and had no effect on the expansion of the live virus gene.Compared with the sensitivity of ordinary qPCR and multiplex PMA-qPCR,it could be seen that the sensitivity of both technologies was 102 copies/μL.The multiplex PMA-qPCR method was used to detect 23 clinical samples,and the accuracy of detecting live viruses was 100%,indicating that the multiplex PMA-qPCR method could effectively distinguish the infectious ASFV in the clinical samples.
作者
卓玛
钱炳旭
吴晓东
戴建君
薛峰
ZHUO Ma;QIAN Bingxu;WU Xiaodong;DAI Jianjun;XUE Feng(MOE Joint International Research Laboratory of Animal Health and Food Safety,Nanjing 210000,China;Sanya Institute of Nanjing Agricultural University,Sanya 572000,China;China Animal Health and Epidemiology Center,Qingdao 266000,China)
出处
《畜牧与兽医》
CAS
北大核心
2023年第4期93-101,共9页
Animal Husbandry & Veterinary Medicine
基金
国家重点研发计划(2021YFD1800500,2020YFA0910200)
海南省科技专项资金(ZDYF2022XDNY248)
江苏省农业科技自主创新基金项目[CX(21)2038]
三亚市南京农业大学研究院指导基金项目(NAUSY-ZD08)。
