啮齿类动物中汉坦病毒感染血清学和病原学检测方法比较研究
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摘要
致病性汉坦病毒的宿主主要为啮齿类动物,其病毒感染状况是人间疫情发生的关键影响因素,可通过检测宿主动物标本中病毒基因组RNA、蛋白抗原及特异性抗体而进行监测。本研究利用367份鼠肺及鼠血标本,对双抗原夹心ELISA(ELISA)、实时荧光RT-PCR(RT-PCR)和免疫荧光(IFA)等三种分别检测抗体、核酸和抗原的方法进行比较评估。ELISA法检出抗体阳性鼠血标本46份,阳性率为12.53%;RT-PCR法检出病毒RNA阳性鼠肺标本28份,阳性率为7.63%;IFA检出抗原阳性鼠肺标本24份,阳性率为6.54%。宿主动物组织标本中检出汉坦病毒RNA和(或)结构蛋白抗原的标本,对应的血液标本中可检出病毒特异性抗体,100%(24/24)IFA检测阳性标本和89.3%(25/28)RT-PCR检测阳性标本对应血标本ELISA抗体检测阳性,反之亦然,检出抗体的标本基本包含了可检出抗原和RNA的标本。RT-PCR与IFA检测结果差异无显著性(χa2=0.64,P>0.05),一致性检验Kappa系数为0.71,一致性高(Z=13.66,P<0.05),首先对血标本开展基于ELISA的特异性抗体检测,可显著缩小RT-PCR或IFA法检测病毒RNA或抗原的范围(χb2=12.04,χc2=20.05,P<0.05)。本研究为宿主动物汉坦病毒感染实验室监测方案优化提供了有益的依据。
Hantavirus infection in host animals(rodents)is a key influencing factor in human epidemics. Such infection can be evaluated by detection of viral genome RNA,antigens,and specific antibodies. We wished to characterize these three methods to optimize laboratory-based procedures to detect hantavirus infection in mice.A total of 367 mice were captured in Jiangxi,Hebei,Liaoning,and Heilongjiang Provinces,China. Lung samples and blood samples from mice were collected. A double-antigen sandwich enzyme-linked immunosorbent assay(ELISA),real-time fluorescent reverse transcription-polymerase chain reaction(RT-PCR),and immunofluorescence assay(IFA)were compared and evaluated. In total,46 blood samples(8.55%)were found to be antibody-positive by the double-antigen sandwich ELISA. Lung samples from 28 mice(4.26%)were found to be positive by real-time fluorescent RT-PCR. Also,24 samples(2.3%)were found to be N protein antigen-positive by IFA. There was no significant difference between RT-PCR and IFA results(χ2 a=0.64,P>0.05),with a Kappa coefficient of 0.71 and good consistency(Z=13.66,P<0.05). All IFA-positive samples(100%,24/24)and most RT-PCR-positive samples(89.3%,25/28)involved antibody detection by ELISA,which is in accordance with the characteristics of infection and replication of the hantavirus in rodents. Most samples detected with antibodies had viral RNA or antigens. Positive samples detected by ELISA were those found to be positive for antigens and RNA. The related detection based on ELISA results could narrow the range of RT-PCR and IFA for detecting viral RNA or antigens significantly[(χ2 b=12.04)(χ2 c=20.05,P<0.05)]. These data provide robust evidence for optimization of the detection procedures for hantavirus surveillance in rodents.
Hantavirus infection in host animals(rodents)is a key influencing factor in human epidemics. Such infection can be evaluated by detection of viral genome RNA,antigens,and specific antibodies. We wished to characterize these three methods to optimize laboratory-based procedures to detect hantavirus infection in mice.A total of 367 mice were captured in Jiangxi,Hebei,Liaoning,and Heilongjiang Provinces,China. Lung samples and blood samples from mice were collected. A double-antigen sandwich enzyme-linked immunosorbent assay(ELISA),real-time fluorescent reverse transcription-polymerase chain reaction(RT-PCR),and immunofluorescence assay(IFA)were compared and evaluated. In total,46 blood samples(8.55%)were found to be antibody-positive by the double-antigen sandwich ELISA. Lung samples from 28 mice(4.26%)were found to be positive by real-time fluorescent RT-PCR. Also,24 samples(2.3%)were found to be N protein antigen-positive by IFA. There was no significant difference between RT-PCR and IFA results(χ2 a=0.64,P>0.05),with a Kappa coefficient of 0.71 and good consistency(Z=13.66,P<0.05). All IFA-positive samples(100%,24/24)and most RT-PCR-positive samples(89.3%,25/28)involved antibody detection by ELISA,which is in accordance with the characteristics of infection and replication of the hantavirus in rodents. Most samples detected with antibodies had viral RNA or antigens. Positive samples detected by ELISA were those found to be positive for antigens and RNA. The related detection based on ELISA results could narrow the range of RT-PCR and IFA for detecting viral RNA or antigens significantly[(χ2 b=12.04)(χ2 c=20.05,P<0.05)]. These data provide robust evidence for optimization of the detection procedures for hantavirus surveillance in rodents.
引文
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[5] Zheng Pang, Aqian Li, Jiandong Li, Jing Qu,Chengcheng He, Shuo Zhang, Chuan Li, Quanfu Zhang,Mifang Liang,and Dexin Li. Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever vi-ruses[J/OL]. PLoS One,2014,9(4):e95635.
[6]张全福,李建东,李伟红,李川,刘琴芝,梁米芳,李德新.双抗原夹心法ELISA在检测抗肾综合征出血热总抗体中的应用[J].中华实验和临床病毒学杂志,2007,21(4):386-388.
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[9] Vaheri A,Strandin T,Hepojoki J,Sironen T,Henttonen H,M?kel?S,Mustonen J. Uncovering the mysteries of hantavirus infections[J]. Nat Rev Microbiol,2013,11(8):539-550.
[10]Kariwa H,Kimura M,Yoshizumi S,Arikawa J,Yoshi-matsu K,Takashima I,Hashimoto N. Modes of Seoul vi-rus infections:persistency in newborn rats and transiency in adult rats[J]. Arch Virol,1996,141(12):2327-2338.
[11]Easterbrook J D,Klein S L. Immunological mechanisms mediating hantavirus persistence in rodent reservoirs[J/OL]. PLoS Pathog,2008,4(11):e1000172.
[12]余鹏博,李慎,魏菁,马长安,卢晓玲,杜水泉,关路媛,郑媛,董建华.直接免疫荧光法和实时荧光定量PCR法检测汉坦病毒效果的比较[J].中华预防医学杂志,2013,47(4):367-370.
[13]南晓伟,宋壮志,韩松,张玉峰,郭卫东. Real time RT-PCR与直接免疫荧光法对鼠肺中汉坦病毒检测效果比较[J].国际病毒学杂志,2016,23(6):401-404.
[14]卢晓玲,郑海潮,李艳红,米宝,马超锋.RT-PCR和直接免疫荧光法检测鼠肺中汉坦病毒结果比较[J].中国卫生检验杂志,2010,(9):2189-2190.
[2] Schmaljohn C S,Hasty S E,Dalrymple J M,LeDuc J W,Lee H W,von Bonsdorff C H,Brummer-Korven-kontio M,Vaheri A,Tsai T F,Regnery H L. Antigen-ic and genetic properties of viruses linked to hemorrhagic fever with renal syndrome[J]. Science,1985,227(4690):1041-1044.
[3]王芹,李建东,张全福,曲靖,王世文. 2014年全国肾综合征出血热监测总结和疫情分析[J].疾病监测,2016,31(3):192-199.
[4] Sch?nrich G,Rang A,Lütteke N,Raftery M J,Char-bonnel N,Ulrich R G. Hantavirus-induced immunity in rodent reservoirs and humans[J]. Immunol Rev,2010,225(1):163-189.
[5] Zheng Pang, Aqian Li, Jiandong Li, Jing Qu,Chengcheng He, Shuo Zhang, Chuan Li, Quanfu Zhang,Mifang Liang,and Dexin Li. Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever vi-ruses[J/OL]. PLoS One,2014,9(4):e95635.
[6]张全福,李建东,李伟红,李川,刘琴芝,梁米芳,李德新.双抗原夹心法ELISA在检测抗肾综合征出血热总抗体中的应用[J].中华实验和临床病毒学杂志,2007,21(4):386-388.
[7]流行性出血热防治手册编写组.流行性出血热防治手册[M].人民卫生出版社,1987:191-199.
[8]梁米芳,宋干,杭长寿,李德新,李盈盈.用单克隆抗体分析流行性出血热病毒的核蛋白抗原位点[J].病毒学报,1989,(1):24-30. DOI:10.13242/j.cnki.bingduxue-bao.000479
[9] Vaheri A,Strandin T,Hepojoki J,Sironen T,Henttonen H,M?kel?S,Mustonen J. Uncovering the mysteries of hantavirus infections[J]. Nat Rev Microbiol,2013,11(8):539-550.
[10]Kariwa H,Kimura M,Yoshizumi S,Arikawa J,Yoshi-matsu K,Takashima I,Hashimoto N. Modes of Seoul vi-rus infections:persistency in newborn rats and transiency in adult rats[J]. Arch Virol,1996,141(12):2327-2338.
[11]Easterbrook J D,Klein S L. Immunological mechanisms mediating hantavirus persistence in rodent reservoirs[J/OL]. PLoS Pathog,2008,4(11):e1000172.
[12]余鹏博,李慎,魏菁,马长安,卢晓玲,杜水泉,关路媛,郑媛,董建华.直接免疫荧光法和实时荧光定量PCR法检测汉坦病毒效果的比较[J].中华预防医学杂志,2013,47(4):367-370.
[13]南晓伟,宋壮志,韩松,张玉峰,郭卫东. Real time RT-PCR与直接免疫荧光法对鼠肺中汉坦病毒检测效果比较[J].国际病毒学杂志,2016,23(6):401-404.
[14]卢晓玲,郑海潮,李艳红,米宝,马超锋.RT-PCR和直接免疫荧光法检测鼠肺中汉坦病毒结果比较[J].中国卫生检验杂志,2010,(9):2189-2190.