CAO Cheng,WEI Mei-sheng,WU Xing-quan,et al.Detection of Bean Pod Mottle Virus RT-PCR Amplicon by Dipstick Assay[J].Soybean Science,2010,29(06):1024-1027.[doi:10.11861/j.issn.1000-9841.2010.06.1024]
菜豆荚斑驳病毒RT-PCR扩增产物胶体金层析检测方法的建立
- Title:
- Detection of Bean Pod Mottle Virus RT-PCR Amplicon by Dipstick Assay
- 文章编号:
- 1000-9841（2010）06-1024-04
- Keywords:
- Colloidal gold; Dipstick; BPMV; RT-PCR ; Nucleic acid detection
- 分类号:
- S565.1
- 文献标志码:
- A
- 摘要:
- 菜豆荚斑驳病毒是我国进境植物检疫性有害生物，为了能在现场快速检测出该病毒，将PCR核酸扩增的高灵敏度和胶体金层析方法快速简便、直观的优点结合起来，建立了PCR扩增产物的胶体金层析检测方法。该方法是用生物素标记上一个引物，用荧光素(或地高辛)标记上另一个引物，通过PCR扩增产生双标记的扩增产物。将兔抗生物素多克隆抗体与20～30 nm大小的胶体金颗粒结合上并固定在释放垫上，同时在硝酸纤维素层析膜上点上抗荧光素(或地高辛)的单克隆抗体作为T检测点，点上羊抗兔多克隆抗体作为C质控点。这种RT-PCR扩增产物可在T点上被检测到，同时C点也要显色。用所建立方法在15 min内可检测出菜豆荚斑驳病毒的RT-PCR扩增产物,免去了溴化乙锭染色和电泳的过程。
- Abstract:
- Bean pod mottle virus (BPMV) is a quarantine pest for China. In order to detect the virus on site, we have developed a rapid method for detection of RT-PCR amplicon of the virus. The method is performed by labeling one prime with biotin the other primer with fluorescein (or digoxigenin). The dual-labeled amplicon can be generated from routine polymerase chain reaction. Rabbit polyclonal antibody against biotin is conjugated with 20~30 nm colloidal gold particles fixed in a conjugate pad.Monoclonal antibody against fluorescein (or digoxigenin) is spotted on nitrocellulose membrane as T dot. Goat anti-rabbit polyclonal antibody is spotted on nitrocellulose membrane as C dot. The RT-PCR amplicon is detected on the test dot(T dot), while the C dot serves as a control. Using this method we have detected the RT-PCR amplicon of bean pod mottle virus within 15 minutes without the staining of ethidium bromide and the running of agarose gel.
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相似文献/References:
[1]张晓雷,檀根甲,魏梅生,等.GICA-RT-PCR检测菜豆荚斑驳病毒的新方法[J].大豆科学,2008,27(06):1019.[doi:10.11861/j.issn.1000-9841.2008.06.1019]
ZHANG Xiao-lei,TAN Gen-jia,WEI Mei-sheng,et al.A New Method for Detecting Bean Pod Mottle Virus by GICA-RT-PCR[J].Soybean Science,2008,27(06):1019.[doi:10.11861/j.issn.1000-9841.2008.06.1019]
备注/Memo
基金项目：国家质量监督检验检疫总局资助项目（2009IK322）。