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本研究采用纳米磁珠提取法(Magnetic Nanoparticles,MNP)、改良的Li Cl沉淀法、CTAB法、TRIzol法和RNeasy Plant M ini Kit 5种方法提取感染啤酒花潜隐类病毒(Hop latent viroid,HLVd)的啤酒花叶片总RNA,结果显示Li Cl沉淀法、CTAB法和M NP法提取的RNA的质量较好,而M NP法具有提取时间短、操作简便,环境友好和批量提取的优势,适用于啤酒花潜隐类病毒RNA的快速提取。体外转录制备HLVd RNA标准品,利用实时荧光定量RT-PCR绘制标准曲线,并对其特异性、灵敏度进行评估。应用纳米磁珠提取啤酒花总RNA,结合实时荧光RT-PCR技术,建立了HLVd的快速、高效的M NP-RT-q PCR定量检测方法。 相似文献
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In order to develop a rapid, sensitive and specific qPCR assay for detection and quantification of Tomato yellow leaf curl virus (TYLCV), a pair of primers and TaqMan probe were designed according to the conserved sequence of known TYLCV isolates. Combining with MNP technique, a novel MNP-qPCR detection method was established and verified based on specificity, sensitivity and reproducibility tests. The results indicated that the Ct value of plotted standard curve showed good linear relationship(R2 =0.9994)with the log of copy number of template. The established method showed a high specificity for TYLCV detection without crossing reaction with Tomato severe leaf curl virus and Tomato yellow leaf curl Sadinia virus, and was 10-fold more sensitive than routine PCR. Both coefficients of variation were less than 2%, indicating a good reproducibility. We have provided a novel method for detection of TYLCV in plant samples rapidly and quantitatively. 相似文献
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应用细菌磁颗粒实时荧光RT-PCR检测南瓜花叶病毒 总被引:1,自引:0,他引:1
A novel real-time RT-PCR method, BMPs based real-time RT-PCR which integrated with magne-tic separation technique of bacterial magnetic particles(BMPs), was set up for detection of Squash mosaic virus(SqMV).After SqMV particles in crude sap were concentrated by BMPs, viral RNAs were released and detected by real time RT-PCR.The results indicated that BMPs based real-time RT-PCR was efficient, and the detection sensibility was equivalent to that of the Trizol based real-time RT-PCR, of which Trizol reagent was used for viral RNAs extration.Comparing to Trizol-based method, the BMPs-based method had advantages of simplicity on operation, time saving for RNA extraction and without using noxious organic chemicals. 相似文献