TaqMan-MGB荧光定量IC/TC-RT-PCR检测菜豆荚斑驳病毒

Detection of Bean Pod Mottle Virus by TaqMan-MGB Real-time IC/TC-RT-PCR

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 regarded as an important quarantine plant pathogen in our country,the risk of BPMV spread from outside increases under the huge soybean import. TaqMan-MGB real-time IC-RT-PCR and TaqMan-MGB real-time TC-RT-PCR methods were developed to detect BPMV by using soybean seed extracts as materials. Based on the conserved region of coat protein sequences of BPMV in Gen Bank,primers and TaqMan-MGB probe were designed. The specificity and sensitivity of these two methods were measured and the comparation of sensitivity of TaqMan-MGB real-time IC-RT-PCR,TaqMan-MGB real-time TC-RT-PCR,IC-RT-PCR and TC-RT-PCR were operated. Two established methods had a high specificity,the comparison results indicated that TC-RT-PCR shared 10- 1crude extracts,IC-RT-PCR and TaqMan-MGB real-time TC-RT-PCR possessed the same sensitivity reaching 10- 3crude extracts while TaqMan-MGB real-time IC-RT-PCR had the highest sensitivity about 10- 5crude extracts. The sensitivity of TaqMan-MGB real-time IC-RT-PCR was 102 times more than TC-RT-PCR,104 times more than TC-RT-PCR. It was practical to use the two established methods in sample detection. So the established TaqMan-MGB real-time IC / TC-RT-PCR method could meet the need of detection with specific capture of antibody or nonspecific capture of tube to catch virion and no need to isdate RNA. It provides the basic approach to detect BPMV in imported soybean seeds with required specificity and sensitivity characteristics.