小反刍兽疫病毒液相芯片检测方法的建立及初步应用
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摘要
为建立小反刍兽疫病毒(PPRV)液相芯片检测方法,根据Gen Bank上公布的PPRV的N蛋白基因序列高度保守区设计引物与探针;对探针进行C12臂修饰后与荧光编码微球偶联,将偶联后的探针与PPRV的PCR产物进行杂交,在Bio-Plex系统上读取荧光信号,建立了小反刍兽疫病毒液相芯片检测方法,并对该方法的特异性和敏感性进行了研究,最终用建立的方法对临床样本进行检测。结果显示,该方法只与PPRV基因的PCR产物反应,而不与其他病毒基因反应,可检出的最低基因拷贝数为5. 78×104copies/m L,表明该方法具有良好的特异性与敏感性。利用该方法对25份临床样本进行检测,共检出阳性样本15份,与传统PCR方法检测结果一致。本研究建立了小反刍兽疫病毒的快速液相芯片检测方法,为进一步建立多种外来动物疫病的液相芯片检测方法奠定了基础。
To establish a simplex suspension array for detecting the peste des petits ruminants virus,the conservative sequence of the PPRV N gene was analyzed,and the specific primers,the probe and the probe coupled with fluorescent microspheres of the N gene were designed and synthesized. Next,the target fragments amplified by asymmetric PCR were hybridized with the C12-modified probe-coupled fluorescent microspheres,and the fluorescent signals were read by the Bio-plex system. The results showed that there were no cross reactions with the other viruses using the established simplex suspension array for PPRV detection. And the detection limit of PPRV was 5. 78×104 copies/m L.These results indicated that the method was successfully established with high specificity and sensitivity. This simplex suspension array method was used to test 25 samples and 15 of them were positive,which was 100% consistent with the conventional PCR method. In conclusion,a simplex suspension array to detect PPRV was successfully established here,laying a foundation for development of a multiplex suspension array for rapid detection of multiple exotic pathogens.
To establish a simplex suspension array for detecting the peste des petits ruminants virus,the conservative sequence of the PPRV N gene was analyzed,and the specific primers,the probe and the probe coupled with fluorescent microspheres of the N gene were designed and synthesized. Next,the target fragments amplified by asymmetric PCR were hybridized with the C12-modified probe-coupled fluorescent microspheres,and the fluorescent signals were read by the Bio-plex system. The results showed that there were no cross reactions with the other viruses using the established simplex suspension array for PPRV detection. And the detection limit of PPRV was 5. 78×104 copies/m L.These results indicated that the method was successfully established with high specificity and sensitivity. This simplex suspension array method was used to test 25 samples and 15 of them were positive,which was 100% consistent with the conventional PCR method. In conclusion,a simplex suspension array to detect PPRV was successfully established here,laying a foundation for development of a multiplex suspension array for rapid detection of multiple exotic pathogens.
引文
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[16]詹爱军,王新卫,卢体康,等. 4种重要虫媒病的核酸液相芯片高通量检测方法的建立[J].畜牧兽医学报,2010,41(2):240-245.
[17]张晓娜. AIV,NDV,IBV和ILTV液相检测芯片方法的建立[D].南京:南京农业大学,2012.
[18]杨显超,吴秀娟,李凯航,等.液相芯片技术同时检测六种猪繁殖障碍病毒的方法研究[J].中国动物检疫,2016,33(12):78-84.
[2]夏咸柱,钱军,杨松涛,等.严把国门,联防联控外来人兽共患病[J].灾害医学与救援(电子版),2014,3(4):204-207.
[3]向蓉,黄勉,向华,等.小反刍兽疫流行状态及检测方法研究进展[J].中国动物检疫,2017(10):46-49.
[4]吴锦艳,尚佑军,田宏,等. 2007—2014年国内外小反刍兽疫流行现状及分析[J].中国兽医学报,2016(04):687-693.
[5]胡瑞,王景林.多重快速鉴别病原微生物的新技术:x MAP液态芯片[J].卫生研究,2007,36(6):759-762.
[6]陈玮.液相芯片技术的原理与应用进展[J].成都医学院学报,2008,3(3):225-231.
[7]程涛,王慧煜,梅琳,等.悬浮芯片技术应用进展[J].生物技术通报,2011(9):48-51.
[8]李林杰,谢军,徐文凯,等.小反刍兽疫病毒的研究进展[J].黑龙江畜牧兽医,2018(1).
[9]李景玉,高玉竹,李元果,等.小反刍兽疫研究进展[J].中国畜牧兽医,2014,41(10):274-280.
[10]陈玮.液相芯片技术的原理与应用进展[J].成都医学院学报,2008,3(3):225-231.
[11] Dunbar S A,Jacobson J W. Quantitative,Multiplexed Detection of Salmonella,and Other Pathogens by Luminexx MAPTMSuspension Array[M]//Salmonella. Humana Press,2007:1-19.
[12] Bortolin S,Black M,Modi H,et al. Analytical validation of the tag-it high-throughput microsphere-based universal array genotyping platform:application to the multiplex detection of a panel of thrombophilia-associated single-nucleotide polymorphisms[J]. Clinical Chemistry,2004,50(11):2028-2036.
[13] Houser B. Bio-Rad's Bio-Plexsuspension array system,x MAP technology overview[J]. Arch Physiol Biochem,2012,118(4):192-196.
[14] Mechaly A,Vitner E,Levy H,et al. Simultaneous Immunodetection of Anthrax,Plague,and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays[J]. J of Cli Micro,2018:JCM.01479-17.
[15] van der Wal F J,Achterberg R P,van Solt-Smits C,et al. Exploring target-specific primer extension in combination with a beadbased suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen[J]. J Vet Diagn Invest,2018,30(1):71-77.
[16]詹爱军,王新卫,卢体康,等. 4种重要虫媒病的核酸液相芯片高通量检测方法的建立[J].畜牧兽医学报,2010,41(2):240-245.
[17]张晓娜. AIV,NDV,IBV和ILTV液相检测芯片方法的建立[D].南京:南京农业大学,2012.
[18]杨显超,吴秀娟,李凯航,等.液相芯片技术同时检测六种猪繁殖障碍病毒的方法研究[J].中国动物检疫,2016,33(12):78-84.