| 240 | 3 | 88 |
| 下载次数 | 被引频次 | 阅读次数 |
建立可检测琉球病毒(Ryukyu virus,RYKV),索尔韦齐病毒(Solwezi virus,SOLV)以及苏里斯病毒(Souris virus,SOUV)等三种沙粒病毒的实时荧光定量RT-PCR检测方法。分析三种病毒流行病学分布特征,从国际公共数据库搜索下载基因组序列,进行比对分析,确定检测靶标,借助primer premier 6.0生物信息学软件,设计引物和探针,建立实时荧光定量RT-PCR检测方法,利用化学合成和体外转录方法制备模拟样本,比较评价三种方法的检测限、特异性、重复性特征。所建实时荧光定量RT-PCR检测方法均可有效扩增检测病毒RNA靶标,检测限分别为40拷贝/μL、7拷贝/μL和15拷贝/μL,检测汉城病毒、汉滩病毒、登革病毒Ⅰ～Ⅳ型分离株、发热伴血小板减少综合病毒及30份健康人血清样本无非特异性扩增,三种病毒相互间以及其他8种出血热相关沙粒病毒RNA间无交叉反应,重复性比较分析显示变异系数均在2%以内。本研究建立的检测RYKV、SOLV和SOUV三种沙粒病毒的实时荧光RT-PCR方法具备用于相关疑似感染者临床样本、宿主动物标本以及进出口物品的筛查检测的潜力,但因未经基于实际病毒感染样本的比较评价,检测结果的解释仍具有一定的局限性。
Abstract：To establish a real-time fluorescent reverse transcription-quantitative polymerase chain reaction(RTqPCR) method for detection of Ryukyu virus(RYKV),Solwezi virus(SOLV)and Souris virus(SOUV)respectively. The epidemiological distribution characteristics of these three viruses were analyzed,and the genomic RNA sequences of these three viruses were collected from public data banks. The detection targets were determined via alignment analysis. Pairs of specific primers and probes were designed using prime premier6.0 software and manual modification. The detection limit,specificities and repeatability of the established methods were evaluated. The established real-time fluorescent RT-q PCR assays can effectively amplify and detect the target viral RNA, with detection limits of 40 copies/μL, 7 copies/μL and 15 copies/μL,respectively. There was no non-specific amplification with Seoul virus,Hantaan virus,dengue virus type Ⅰ ～Ⅳ isolates,SFTS virus and the sera of 30 healthy adults. Cross reaction between the three viruses and with the RNA of other eight hemorrhagic fever related arenavirus was not detected. The analysis of repeatability showed that the coefficient of variation was within 2%. The real-time RT-PCR methods for detection of RYKV,SOLV and SOUV could be used for the screening of clinical samples,host animal samples and related suspected infected persons.
[1]Sarute N,Ross S R.New World arenavirus biology[J].Annu Rev Virol,2017,4(1):141-158.
[2]Radoshitzky S R,Buchmeier M J,Charrel R N,Clegg J C S,Gonzalez J J,Günther S,Hepojoki J,Kuhn J H,Lukashevich I S,Romanowski V,Salvato M S,Sironi M,Stenglein M D,De La Torre J C.ICTV Virus Taxonomy Profile:Arenaviridae[J].J Gen Virol,2019,100(8):1200-1201.
[3]Sarute N,Ross S R.New World Arenavirus Biology[J].Annu Rev Virol,2017,4(1):141-158.
[4]Radoshitzky S R,Bào Y,Buchmeier M J,Charrel RN,Clawson A N,Clegg C S,De Risi J L,Emonet S,Gonzalez J P,Kuhn J H,Lukashevich I S,Peters C J,Romanowski V,Salvato M S,Stenglein M D,De La Torre J C.Past,present,and future of arenavirus taxonomy[J].Arch Virol,2015,160(7):1851-1874.
[5]Charrel R N,Lamballerie X D.Zoonotic aspects of arenavirus infections[J].Vet Microbiol,2010,140(3):213-220.
[6]Brisse M E,Ly H.Hemorrhagic Fever-causing arenaviruses:lethal pathogens and potent immune suppressors[J].Front Immunol,2019,10(372):2.
[7]Fedeli C,Moreno H,Kunz S.Novel insights into cell entryof emerging human pathogenic arenaviruses[J].JMol Bio,2018,430(13):1839-1852.
[8]Lozano M E,Posik D M,Albari?o C G,Schujman G,Ghiringhelli P D,Calderón G,Sabattini M,Romanowski V.Characterization of arenaviruses using a family-specific primer set for RT-PCR amplification and RFLP analysis Its potential use for detection of uncharacterized arenaviruses[J]Virus Res,1997,49(1):79-89.
[9]Pang Z,Li A,Li J,Qu J,He C,Zhang S,Li C,Zhang Q,Liang M,Li D.Comprehensive Multiplex One-Step Real-Time Taq Man q RT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses[J/OL].PLo S One,2014,9(4):1-10.
[10]Wu Z,Lu L,Du J,Yang L,Ren X,Liu B,Jiang J,Yang J,Dong J,Sun L,Zhu Y,Li Y,Zheng D,Zhang C,Su H,Zheng Y,Zhou H,Zhu G,Li H,Chmura A,Yang F,Daszak P,Wang J,Liu Q,Jin Q.Comparative analysis of rodent and small mammal viromes to better understand the wildlife origin of emerging infectious diseases[J].Medsci,2018,6(1):1-14.
[11]Grajkowska L T,Pedras-Vasconcelos J A,Sauder C,Verthelyi D,Puig M.High-throughput real-time PCRfor early detection and quantitation of arenavirus Tacaribe[J].J Virol Methods,2009,159 (2):239-243.
[12]Ly H.Differential immune responses to new world and old world mammalian arenaviruses[J].Int J Mol Sci,2017,18(5):1040.
[13]Kerber R,Reindl S,Romanowski V,Gómez R M,Ogbaini-Emovon E,Günther S,ter Meulen J.Research efforts to control highly pathogenic arenaviruses:Asummary of the progress and gaps[J].J Clin Virol,2015,64:120-127.
[14]Cordey S,Sahli R,Moraz M L,Estrade C,Morandi L,Cherpillod P,Charrel RN,Kunz S,Kaiser L.Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay[J].J Virol Methods,2011,177(1):118-122.
[15]Olschl?ger S,Lelke M,Emmerich P,Panning M,Drosten C,Hass M,Asogun D,Ehichioya D,Omilabu S,Günther S.Improved detection of lassa virus by reverse transcription-pcr targeting the 5 region of SRNA[J].J Clin Microbiol,2010,48(6):2009-2013.
[16]Klaus J P,Botten J.Highly sensitive assay for measurement of arenavirus-cell attachment[J].J Vis Exp,2016,2(109):1-9.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.003979
中图分类号:R373
引用信息:
[1]杜珊珊,赖丽金,李阿茜,等.琉球病毒、索尔韦齐病毒、苏里斯病毒等三种沙粒病毒实时荧光RT-PCR检测方法研究[J].病毒学报,2021,37(04):809-815.DOI:10.13242/j.cnki.bingduxuebao.003979.
基金信息:
重大传染病防治专项：（项目号：2018ZX10711001,2018ZX10101-002,2016ZX10004222-002）题目：病毒感染高通量快速检测与应急筛检技术研究; 非洲病毒性出血热实验室检测及血清流行病学调查研究：国家科技重大专项~~
2021-05-26
2021-05-26
2021-05-26