狂犬病病毒TaqMan qRT-PCR检测方法的建立
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摘要
建立狂犬病病毒TaqMan qRT-PCR检测方法,并进行评价和初步应用。针对狂犬病病毒L基因保守区域设计特异性引物、探针,建立PCR反应体系并评价检测体系的扩增效率、灵敏度、特异性和准确性等指标,灵敏度评价结果与现有巢式RT-PCR和基于N基因qRT-PCR检测方法相比较。结果显示本研究建立的基于L基因qRTPCR检测体系扩增效率高于90%;最小检测病毒滴度为2.7×10~(-3)FFU/mL,灵敏度高于基于N基因qRT-PCR和巢式RT-PCR 100倍;最小检测核酸拷贝数为100拷贝,建立了基于目的基因RNA标准品的标准曲线,可用于定量检测;对于我国的狂犬病病毒流行毒株检测范围较广,动物样本检测结果与金标准DFA检测结果相一致,准确性较高。本研究建立的狂犬病病毒qRT-PCR可用于狂犬病病毒实验室检测。
A rapid and sensitive real-time RT-PCR assay for detection of rabies virus based on polymerase gene was established and evaluated.Specific primers and probes were designed based on the conserved region of L gene of rabies virus.The amplification efficiency,sensitivity,specificity and accuracy of the detection system were evaluated.The results of sensitivity were compared with the conventional nested RTPCR and TaqMan qRT-PCR assay based on nucleoprotein gene which were reported before.The results showed that the L gene real-time PCR assay established in this study was negative for JEV,DENV and other 9 lyssaviruses except RABV.The limit of detection of viral titer was 2.7×10~(-3) FFU/mL which was 102 times lower than the conventional nested RT-PCR and the N gene real-time RT-PCR assay.The limit of detection of RNA standard copy number was 100 copies.A standard curve based on the RNA standard of the target gene was established and can be used for quantitative detection.The established TaqMan qRT-PCR assay had a wide range of detection result for epidemic rabies virus strains in China.Several animal samples were collected and detected,and the detection results were consistent with the gold standard DFA.The established TaqMan qRT-PCR assay for detection of rabies virus can be applied to laboratory detection of rabies virus.
A rapid and sensitive real-time RT-PCR assay for detection of rabies virus based on polymerase gene was established and evaluated.Specific primers and probes were designed based on the conserved region of L gene of rabies virus.The amplification efficiency,sensitivity,specificity and accuracy of the detection system were evaluated.The results of sensitivity were compared with the conventional nested RTPCR and TaqMan qRT-PCR assay based on nucleoprotein gene which were reported before.The results showed that the L gene real-time PCR assay established in this study was negative for JEV,DENV and other 9 lyssaviruses except RABV.The limit of detection of viral titer was 2.7×10~(-3) FFU/mL which was 102 times lower than the conventional nested RT-PCR and the N gene real-time RT-PCR assay.The limit of detection of RNA standard copy number was 100 copies.A standard curve based on the RNA standard of the target gene was established and can be used for quantitative detection.The established TaqMan qRT-PCR assay had a wide range of detection result for epidemic rabies virus strains in China.Several animal samples were collected and detected,and the detection results were consistent with the gold standard DFA.The established TaqMan qRT-PCR assay for detection of rabies virus can be applied to laboratory detection of rabies virus.
引文
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[7]李浩,陶晓燕,宋淼,张强,莫兆军,周开姣,张红,戴德芳,王定明,周金柱,唐青,梁国栋.狂犬病高发地区犬只感染情况调查分析[J].中华实验和临床病毒学杂志,2008,22(3):161-164.
[8]周兰贞,刘永娣,戴美兰,容伟华,王红冰,田华,张剑平.细胞荧光转化灶法检测狂犬病病毒滴度[J].中国生物制品学杂志,2015,28(10):1092-1096.
[9]王梦舒,郑宇,郭丽姝,陶雷,程晓耕,徐菲.直接免疫荧光法检测狂犬病病毒滴度条件的优化及验证[J].中国生物制品学杂志,2014,27(11):1477-1480.
[10]周康凤,姜立民,黄海鹰,毛子安,陈秋红.间接免疫荧光技术检测狂犬病毒感染性滴度的可行性研究[J].国际流行病学传染病学杂志,2006,33(6):365-367.
[11]Tao X Y,Tang Q,Rayner S,Guo Z Y,Li H,Lang S L,Yin C P,Han N,Fang W,Adams J,Song M,Liang G D.Molecular phylodynamic analysis indicates lineage displacement occurred in Chinese rabies epidemics between 1949to 2010[J/OL].PLoS Negl Trop Dis,2013,7(7):e2294.
[12]Li H,Guo Z Y,Zhang J,Tao X Y,Zhu W Y,Tang Q,Liu H T.Whole genome sequencing and comparisons of different Chinese rabies virus lineages including the first complete genome of an Arctic-like strain in China[J].Biomed Environ Sci,2016,29(5):340-346.
[13]Guo Z,Tao X,Yin C,Han N,Yu J,Li H,Liu H,Fang W,Adams J,Wang J,Liang G,Tang Q,Rayner S.National borders effectively halt the spread of rabies:The current rabies epidemic in China is dislocated from cases in neighboring countries[J/OL].PLoS Negl Trop Dis,2013,7(1):e2039.
[14]Tang Q,Orciari L A,Rupprechti C E,Zhao X Q,Li Z G,Yang W S.Sequencing and position analysis of the glycoprotein gene of four Chinese rabies viruses[J].Virol Sin,2000,15(1):22-33.
[15]He C Q,Meng S L,Yan H Y,Ding N Z,He H B,Yan J X,Xu G L.Isolation and identification of a novel rabies virus lineage in China with natural recombinant nucleoprotein gene[J/OL].PLoS One,2012,7(12):e49992.
[16]Zhang J,Zhang H L,Tao X Y,Li H,Tang Q,Jiang X Y,Liang G D.The full-length genome analysis of a street rabies virus strain isolated in Yunnan province of China[J].Virol Sin,2012,27(3):204-213.
[17]Wang L,Liu Y,Zhang S,Wang Y,Zhao J,Miao F,Hu R.A SYBR-green I quantitative real-time reverse transcription-PCR assay for rabies viruses with different virulence.[J].Virol Sin,2014,29(2):131-132.
[18]张强,唐青,刘卫滨,李浩,梁国栋.狂犬病毒TaqMan PCR检测方法的建立[J].中华流行病学杂志,2006,27(10):889-893.
[19]许运斌,邵明富,范金红,孙彦伟,席进,涂长春.基因1型狂犬病病毒一步法荧光定量RT-PCR检测方法的建立[J].中国人兽共患病学报,2011,27(4):297-302.
[20]周航,李昱,陈瑞丰,陶晓燕,于鹏程,曹守春,李丽,陈志海,朱武洋.狂犬病预防控制技术指南(2016版)[J].中华流行病学杂志,2016,37(2):139-163.
[21]Wadhwa A,Wilkins K,Gao J,Condori Condori RE,Gigante CM,Zhao H,Ma X,Ellison JA,Greenberg L,Velasco-Villa A,Orciari L,Li Y.A pan-lyssavirus Taqman real-time RT-PCR assay for the detection of highly variable rabies virus and other lyssaviruses[J/OL].PLoS Negl Trop Dis,2017,11(1):e0005258.
[2]Wang L,Tang Q,Liang G.Rabies and rabies virus in wildlife in mainland China,1990-2013[J].I Int J Infect Dis,2014,25:122-129.
[3]李艳荣,祝丽玲,朱武洋,陶晓燕.中国2016年狂犬病流行病学特征分析[J].中华流行病学杂志,2018,39(1):40-43.
[4]Jackson A C,Warrell M J,Rupprecht C E,Ertl H C,Dietzschold B,O′Reilly M,Leach R P,Fu Z F,Wunner W H,Bleck T P,Wilde H.Management of rabies in humans.[J].Clin Infect Dis,2003,36(1):60-63.
[5]Wakeley P R,Johnson N,McElhinney L M,Marston D,Sawyer J,Fooks A R.Development of a real-time,differential RT-PCR TaqMan assay for lyssavirus genotypes 1,5 and 6[J].Dev Biol,2006,126(126):227-236.
[6]Nagaraj T,Vasanth J P,Desai A,Kamat A,Madhusudana S N,Ravi V.Ante mortem diagnosis of human rabies using saliva samples:comparison of real time and conventional RT-PCR techniques.[J].J Clin Virol,2006,36(1):17-23.
[7]李浩,陶晓燕,宋淼,张强,莫兆军,周开姣,张红,戴德芳,王定明,周金柱,唐青,梁国栋.狂犬病高发地区犬只感染情况调查分析[J].中华实验和临床病毒学杂志,2008,22(3):161-164.
[8]周兰贞,刘永娣,戴美兰,容伟华,王红冰,田华,张剑平.细胞荧光转化灶法检测狂犬病病毒滴度[J].中国生物制品学杂志,2015,28(10):1092-1096.
[9]王梦舒,郑宇,郭丽姝,陶雷,程晓耕,徐菲.直接免疫荧光法检测狂犬病病毒滴度条件的优化及验证[J].中国生物制品学杂志,2014,27(11):1477-1480.
[10]周康凤,姜立民,黄海鹰,毛子安,陈秋红.间接免疫荧光技术检测狂犬病毒感染性滴度的可行性研究[J].国际流行病学传染病学杂志,2006,33(6):365-367.
[11]Tao X Y,Tang Q,Rayner S,Guo Z Y,Li H,Lang S L,Yin C P,Han N,Fang W,Adams J,Song M,Liang G D.Molecular phylodynamic analysis indicates lineage displacement occurred in Chinese rabies epidemics between 1949to 2010[J/OL].PLoS Negl Trop Dis,2013,7(7):e2294.
[12]Li H,Guo Z Y,Zhang J,Tao X Y,Zhu W Y,Tang Q,Liu H T.Whole genome sequencing and comparisons of different Chinese rabies virus lineages including the first complete genome of an Arctic-like strain in China[J].Biomed Environ Sci,2016,29(5):340-346.
[13]Guo Z,Tao X,Yin C,Han N,Yu J,Li H,Liu H,Fang W,Adams J,Wang J,Liang G,Tang Q,Rayner S.National borders effectively halt the spread of rabies:The current rabies epidemic in China is dislocated from cases in neighboring countries[J/OL].PLoS Negl Trop Dis,2013,7(1):e2039.
[14]Tang Q,Orciari L A,Rupprechti C E,Zhao X Q,Li Z G,Yang W S.Sequencing and position analysis of the glycoprotein gene of four Chinese rabies viruses[J].Virol Sin,2000,15(1):22-33.
[15]He C Q,Meng S L,Yan H Y,Ding N Z,He H B,Yan J X,Xu G L.Isolation and identification of a novel rabies virus lineage in China with natural recombinant nucleoprotein gene[J/OL].PLoS One,2012,7(12):e49992.
[16]Zhang J,Zhang H L,Tao X Y,Li H,Tang Q,Jiang X Y,Liang G D.The full-length genome analysis of a street rabies virus strain isolated in Yunnan province of China[J].Virol Sin,2012,27(3):204-213.
[17]Wang L,Liu Y,Zhang S,Wang Y,Zhao J,Miao F,Hu R.A SYBR-green I quantitative real-time reverse transcription-PCR assay for rabies viruses with different virulence.[J].Virol Sin,2014,29(2):131-132.
[18]张强,唐青,刘卫滨,李浩,梁国栋.狂犬病毒TaqMan PCR检测方法的建立[J].中华流行病学杂志,2006,27(10):889-893.
[19]许运斌,邵明富,范金红,孙彦伟,席进,涂长春.基因1型狂犬病病毒一步法荧光定量RT-PCR检测方法的建立[J].中国人兽共患病学报,2011,27(4):297-302.
[20]周航,李昱,陈瑞丰,陶晓燕,于鹏程,曹守春,李丽,陈志海,朱武洋.狂犬病预防控制技术指南(2016版)[J].中华流行病学杂志,2016,37(2):139-163.
[21]Wadhwa A,Wilkins K,Gao J,Condori Condori RE,Gigante CM,Zhao H,Ma X,Ellison JA,Greenberg L,Velasco-Villa A,Orciari L,Li Y.A pan-lyssavirus Taqman real-time RT-PCR assay for the detection of highly variable rabies virus and other lyssaviruses[J/OL].PLoS Negl Trop Dis,2017,11(1):e0005258.