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菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)是我国重要的检疫性有害生物,BPMV传入的风险随大豆进境数量增多而加大。本文针对菜豆荚斑驳病毒,以大豆种子提取液为材料,分别建立了TaqMan-MGB荧光定量IC-RTPCR和TaqMan-MGB荧光定量TC-RT-PCR快速检测方法。根据GenBank公布的BPMV外壳蛋白基因序列,选择其保守区域,设计1对特异性引物和1条TaqMan-MGB探针,测定了TaqMan-MGB荧光定量IC-RT-PCR和TaqMan-MGB荧光定量TC-RT-PCR方法的特异性和灵敏度,并将TaqMan-MGB荧光定量IC-RT-PCR、TaqMan-MGB荧光定量TC-RTPCR、IC-RT-PCR和TC-RT-PCR 4种检测方法的灵敏度进行比较。结果表明:所建立的两种检测方法特异性良好;TCRT-PCR的灵敏度为10-1倍病毒提取液原液;IC-RT-PCR和TaqMan-MGB荧光定量TC-RT-PCR灵敏度相当,为10-3倍病毒提取液原液;TaqMan-MGB荧光定量IC-RT-PCR灵敏度最高,为10-5倍病毒提取液原液,是IC-RT-PCR和TaqMan-MGB荧光定量TC-RT-PCR检测方法的102倍,是TC-RT-PCR检测方法的104倍;两种检测方法均能较好应用于实际大豆样品的检测。本文所建立的TaqMan-MGB荧光定量IC/TC-RT-PCR检测方法利用抗原特异性或试管非特异性捕捉病毒粒子,无需提取RNA,为进境大豆种子上BPMV的检测提供了稳定、快速、简便的技术依据,其较高的灵敏度和特异性具有较好的应用价值。
Bean pod mottle virus (BPMV) is an important quarantine pest in our country. The risk of BPMV introduction increases with the increase of the number of soybean entering. In this paper, the rapid detection method of TaqMan-MGB fluorescent quantitative IC-RTPCR and TaqMan-MGB fluorescence quantitative TC-RT-PCR was established for the bean pod mottle virus using the soybean seed extract as the material. According to the sequence of BPMV coat protein published in GenBank, we selected one conserved region and designed one pair of specific primers and one TaqMan-MGB probe. The TaqMan-MGB fluorescent quantitative IC-RT-PCR and TaqMan-MGB fluorescent quantitative TC- RT-PCR method and the sensitivity of TaqMan-MGB fluorescence quantitative IC-RT-PCR, TaqMan-MGB fluorescence quantitative TC-RTPCR, IC-RT-PCR and TC- Compare The results showed that the specificity of the two detection methods was good. The sensitivity of TCRT-PCR was 10-1 times of that of virus extract. The sensitivity of TC-RT-PCR and TaqMan-MGB fluorescence quantitative PCR-RT-PCR was 10 -3 times the virus extract liquid; TaqMan-MGB fluorescence quantitative IC-RT-PCR highest sensitivity of 10-5 times the virus extract liquid, is IC-RT-PCR and TaqMan-MGB fluorescence quantitative TC-RT-PCR detection method Of the 102 times, is TC-RT-PCR detection of 104 times; both detection methods can be better applied to the actual detection of soybean samples. The TaqMan-MGB fluorescence quantitative IC / TC-RT-PCR detection method established in this paper utilizes antigen-specific or non-specific capture of virus particles in a tube without the need for RNA extraction. It provides a stable, rapid and simple method for the detection of BPMV on the incoming soybean seeds The technical basis of its high sensitivity and specificity have good application value.