小鼠诺如病毒逆转录重组酶聚合酶扩增检测方法的初步建立
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摘要
小鼠是科学研究中使用最多的实验动物,小鼠诺如病毒(MNV)是试验小鼠感染率最高的病毒性病原,因此MNV感染小鼠将影响研究结果,及时对小鼠健康状态进行监测必不可少。本研究根据逆转录重组酶聚合酶扩增技术(RT-RPA),通过引物探针的筛选,建立了检测MNV的RT-RPA方法。该方法具有较好的敏感性和特异性,通过荧光扩增曲线判定样本中是否含有MNV,在20 min内即可实现样本的检测。将该方法应用于临床样本的检测,结果显示32个样本中有6个阳性,和RT-qPCR检测结果总符合率达到97%。该方法的建立对于试验小鼠的日常病原监测和MNV流行病学研究具有重要意义。
The infection rate of the murine norovirus in mice is the highest among the murine viral pathogens.The mice are the most widely used laboratory animals in scientific research,thus the mice that infected with MNV may resulted in the mistake of the research.Monitoring the healthy status of the mice is vital important.In the present study,a reverse transcription recombinase polymerase amplification assay(RT-RPA) was developed via screening the primers and probes.The RT-RPA for detecting MNV had good sensitivity and specificity,the positive samples were determined by the generation of amplification curve in 20 minutes.The RT-RPA was used to detect the clinical samples and the result revealed that 6 positive samples were detected in 32 samples.The coincidence rate between RT-RPA and RT-qPCR reached 97%.The developed RT-RPA played an important role in routine monitoring and epidemiology of MNV in laboratory mice.
The infection rate of the murine norovirus in mice is the highest among the murine viral pathogens.The mice are the most widely used laboratory animals in scientific research,thus the mice that infected with MNV may resulted in the mistake of the research.Monitoring the healthy status of the mice is vital important.In the present study,a reverse transcription recombinase polymerase amplification assay(RT-RPA) was developed via screening the primers and probes.The RT-RPA for detecting MNV had good sensitivity and specificity,the positive samples were determined by the generation of amplification curve in 20 minutes.The RT-RPA was used to detect the clinical samples and the result revealed that 6 positive samples were detected in 32 samples.The coincidence rate between RT-RPA and RT-qPCR reached 97%.The developed RT-RPA played an important role in routine monitoring and epidemiology of MNV in laboratory mice.
引文
[1] Karst S M,WOBUS C E,LAY M,et al.STAT1-dependent innate immunity to a norwalk-like virus[J].Science,2003,299 (5612):1575-1578.
[2] WOBUS C E,KARST S M,THACKRAY L B,et al.Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages[J].PLoS Biol,2004,2(12):e432.
[3] 王翠娥,陈立超,周倩,等.实验大鼠和小鼠多种病毒的血清学检测结果分析[J].实验动物科学,2014,31 (2),20-24.
[4] PAIK J,FIERCE Y,DRIVDAHL,et al.Effects of murine norovirus infection on a mouse model of diet-induced obesity and insulin resistance[J].Comp Med,2010,60 (3):189-195.
[5] 袁文,王静,赵维波,等.结合内标的小鼠诺如病毒荧光定量RT-PCR检测方法的建立及应用[J].中国实验动物学报,2015,23 (1):49-56.
[6] PIEPENBURG O,WILLIAMS C H,STEMPLE D,et al.DNA detection using recombination proteins[J].PLoS Biol,2006,4 (7):e204.
[7] YEHIA N,ARAFA A S,ABD EL WAHED A,et al.Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection[J].J Virol Methods,2015,223:45-49.
[8] YANG Y,QIN X,ZHANG W,et al.Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus[J].Mol Cell Probes,2017,33:32-35.
[9] YANG Y,QIN X,SUN Y,et al.Development of isothermal recombinase polymerase amplification assay for rapid detection of porcine circovirus type 2[J].Biomed Res Int,2017(2):1-8.
[10] SHAHIN K,GUSTAVO RAMIREZ-PAREDES J,HAROLD G,et al.Development of a recombinase polymerase amplification assay for rapid detection of Francisella noatunensis subsp.orientalis[J].PLoS One,2018,13 (2):e0192979.
[11] 袁文,张谱华,王静,等.小鼠诺如病毒分离鉴定及病毒衣壳蛋白基因序列分析[J].中国实验动物学报,2013,21 (6):18-27.
[12] 潘金春,赵维波,陈梅玲,等.2013年-2015年广东地区实验小鼠和大鼠微生物学及寄生虫学调查[J].中国比较医学杂志,2017,27 (2):64-69.
[13] NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[14] MOORE M D,JAYKUS L A.Development of a recombinase polymerase amplification assay for detection of epidemic human noroviruses[J].Sci Rep,2017(7):40244.
[15] FAYE O,SOROPOGUI B.Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015[J].Euro Surveill,2015,20:44.
[2] WOBUS C E,KARST S M,THACKRAY L B,et al.Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages[J].PLoS Biol,2004,2(12):e432.
[3] 王翠娥,陈立超,周倩,等.实验大鼠和小鼠多种病毒的血清学检测结果分析[J].实验动物科学,2014,31 (2),20-24.
[4] PAIK J,FIERCE Y,DRIVDAHL,et al.Effects of murine norovirus infection on a mouse model of diet-induced obesity and insulin resistance[J].Comp Med,2010,60 (3):189-195.
[5] 袁文,王静,赵维波,等.结合内标的小鼠诺如病毒荧光定量RT-PCR检测方法的建立及应用[J].中国实验动物学报,2015,23 (1):49-56.
[6] PIEPENBURG O,WILLIAMS C H,STEMPLE D,et al.DNA detection using recombination proteins[J].PLoS Biol,2006,4 (7):e204.
[7] YEHIA N,ARAFA A S,ABD EL WAHED A,et al.Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection[J].J Virol Methods,2015,223:45-49.
[8] YANG Y,QIN X,ZHANG W,et al.Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus[J].Mol Cell Probes,2017,33:32-35.
[9] YANG Y,QIN X,SUN Y,et al.Development of isothermal recombinase polymerase amplification assay for rapid detection of porcine circovirus type 2[J].Biomed Res Int,2017(2):1-8.
[10] SHAHIN K,GUSTAVO RAMIREZ-PAREDES J,HAROLD G,et al.Development of a recombinase polymerase amplification assay for rapid detection of Francisella noatunensis subsp.orientalis[J].PLoS One,2018,13 (2):e0192979.
[11] 袁文,张谱华,王静,等.小鼠诺如病毒分离鉴定及病毒衣壳蛋白基因序列分析[J].中国实验动物学报,2013,21 (6):18-27.
[12] 潘金春,赵维波,陈梅玲,等.2013年-2015年广东地区实验小鼠和大鼠微生物学及寄生虫学调查[J].中国比较医学杂志,2017,27 (2):64-69.
[13] NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[14] MOORE M D,JAYKUS L A.Development of a recombinase polymerase amplification assay for detection of epidemic human noroviruses[J].Sci Rep,2017(7):40244.
[15] FAYE O,SOROPOGUI B.Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015[J].Euro Surveill,2015,20:44.