[1]刘立兵?,王金凤?,张若曦,等.口蹄疫病毒实时荧光RT-RPA快速检测方法的建立[J].中国预防兽医学报,2019,(02):168-173.[doi:0.3969/j.issn.1008-0589.201805023]
 LIU Li-bing,WANG Jin-feng,ZHANG Ruo-xi,et al.Development of the real-time reverse transcription recombinase polymerase amplification for rapid detection of foot-and-mouth disease virus[J].Chinese journal of preventive veterinary medicine,2019,(02):168-173.[doi:0.3969/j.issn.1008-0589.201805023]
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口蹄疫病毒实时荧光RT-RPA快速检测方法的建立()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年02
页码:
168-173
栏目:
诊断和检测技术
出版日期:
2019-02-15

文章信息/Info

Title:
Development of the real-time reverse transcription recombinase polymerase amplification for rapid detection of foot-and-mouth disease virus
文章编号:
1008-0589(2019)02-0168-06
作者:
 

刘立兵12?王金凤12?张若曦3石蕊寒12韩庆安3李睿文4王建昌12*袁万哲4*

 (1. 河北省检验检疫科学技术研究院,河北 石家庄 050051;2. 河北出入境检验检疫局技术中心,河北 石家庄 050051;
3. 河北省动物疫病预防控制中心,河北 石家庄 050035;4. 河北农业大学 动物医学院,河北 保定 071001)
Author(s):
 

LIU Li-bing112? WANG Jin-feng12? ZHANG Ruo-xi3 SHI Rui-han12 HAN Qing-an3 LI Rui-wen4 WANG Jian-chang12* YUAN Wan-zhe4*

 (1. Hebei Academy of Science and Technology for Inspection and Quarantine, Shijiazhuang 050051, China; 2. Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang 050051, China; 3. Hebei Animal Disease Control Center, Shijiazhuang 050035, China; 4. College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China)
关键词:
口蹄疫病毒3D 基因exo探针实时荧光RT-RPA
Keywords:
foot-and-mouth disease virus  3D gene  exo probe  real-time RT-RPA
分类号:
S852.65
DOI:
0.3969/j.issn.1008-0589.201805023
文献标志码:
A
摘要:
为建立一种简单、快速、特异的口蹄疫病毒(FMDV)分子检测方法,本研究基于FMDV的3D基因,设计特异性引物和exo探针,建立了用于FMDV快速检测的等温实时荧光反转录重组酶聚合酶(Real-time RT-RPA)方法。该方法采用便携式等温扩增仪-Genie III实时监测扩增结果,40 ℃ 20 min即可完成检测。结果显示,FMDV RT-RPA方法能够特异性扩增FMDV 3D基因,而对水泡性口炎病毒、猪繁殖与呼吸综合征病毒、猪瘟病毒、脑心肌炎病毒等均无扩增;以体外转录的FMDV 3D RNA作为模板,该方法在95 %置信区间检出下限为1.0×102拷贝/μL,组内和组间变异系数均小于1 %;使用43份含有灭活FMDV的模拟临床样品分析显示,RT-RPA对FMDV的检出率为34.9 % (15/43),略低于荧光定量RT-PCR的检出率(39.5 %,17/43),而两种方法的符合率为95.3 % (41/43)。RT-RPA能够在6 min~16 min内完成检测,而实时荧光RT-PCR则需要30 min~51 min。本研究所建立的实时荧光RT-RPA方法反应快速,特异性强,灵敏性高,操作简单,结合便携式具有荧光检测功能的等温扩增仪Genie III,组建了FMDV快速检测平台,在基层兽医部门,尤其在野外及疫情现场的FMDV检测中具有极大的应用潜力,为设备仪器有限的实验室和田间对FMDV的快速检测提供了一种有效的方法,对于条件有限地区FMD的防控具有重要意义。
  
Abstract:
To develop a simple, rapid and specific method for the foot-and-mouth disease virus (FMDV) detection, the real-time reverse-transcription recombinase polymerase amplification assay (real-time RT-RPA) was developed using primers targetting the conserved region of the 3D gene, and the amplification was performed at 40℃ in the portable Genie III scanner device for 20 min under the real-time monitoring. The amplification of real-time RT-RPA was specific for FMDV, but not for VSV, PRRSV, CSFV, EMCV, PRV and PCV2, displaying a high specificity. Using the in vitro transcribed FMDV RNA as template, the analytical sensitivity was 102 copies at 95% cases. The co-efficient of variations in intra- and inter-assays were both less than 1%. The assay performance was evaluated by testing 43 clinical samples by the real-time RT-RPA and a real-time RT-PCR assay. Fifteen samples were FMDV positive in RT-RPA (34.9%, 15/43), while the positive rate was 39.5% (17/43) in real-time RT-PCR. The diagnostic agreement between the two assays was 95.3% (41/43). It took 6-16 min in the RT-RPA assay to obtain the positive results, while the real-time RT-PCR took about 30 min to 51 min. The developed FMDV RT-RPA assay is rapid, highly specific, highly sensitive and easy to perform, and the FMDV rapid detection platform was established with Genie III, which provides a rapid and reliable diagnostic tool for FMD in the field, and is of great significance in FMD provention.

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(本文编辑:李 爽)

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更新日期/Last Update: 2019-03-06