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首页> 外文期刊>Plant Disease >A Rapid, Sensitive Assay for Ralstonia solanacearum Race 3 Biovar 2 in Plant and Soil Samples Using Magnetic Beads and Real-Time PCR
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A Rapid, Sensitive Assay for Ralstonia solanacearum Race 3 Biovar 2 in Plant and Soil Samples Using Magnetic Beads and Real-Time PCR

机译：利用磁珠和实时PCR快速，灵敏地测定植物和土壤样品中青枯雷尔氏菌种族3 Biovar 2

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Ha, Y., Kim, J.-S., Denny, T. P., and Schell, M. A. 2012. A rapid, sensitive assay for Ralstonia solanacearum race 3 biovar 2 in plant and soil samples using magnetic beads and real-time PCR. Plant Dis. 96:258-264. The Ralstonia solanacearum species complex causes economically significant diseases in many plant families worldwide. Although generally limited to the tropics and subtropics, strains designated race 3 biovar 2 (R3Bv2) cause disease in cooler tropical highlands and temperate regions. R3Bv2 has not become established in North America but, due to concerns that it could devastate the U.S. potato industry, it has been designated a Select Agent, and is subject to strict quarantine regulations. Quarantine screening for R3Bv2 requires rapid and robust assays applicable to small populations present in plant tissues or soil, and must distinguish R3Bv2 from the multiple other R. solanacearum subgroups. We developed a 100%-accurate real-time polymerase chain reaction (RT-PCR) assay that can detect R3Bv2 populations >1,000 cells ml(-1). However, detection by RT-PCR was inhibited by compounds present in some plant and soil samples. Therefore, we developed simple immunomagnetic separation (IMS) and magnetic capture hybridization (MCH) methods to purify R. solanacearum cells or DNA from PCR inhibitors. When coupled with RT-PCR, these tools permitted detection of R3Bv2 at levels >500 cells ml(-1) in stem, tuber, and soil samples when direct RT-PCR failed, and reduced detection time from days to hours. IMS-RT-PCR was usually more sensitive than MCH-RT-PCR, especially at lower population levels.
机译：Ha，Y.，Kim，J.-S.，Denny，T. P.和Schell，M.A.2012。使用磁珠和实时PCR对植物和土壤样品中的茄形青枯菌种族3 biovar 2进行快速，灵敏的测定。植物病96：258-264。 Ralstonia solanacearum种复合物在世界范围内的许多植物科中引起具有经济意义的疾病。尽管通常限于热带和亚热带，但指定为第3种biovar 2（R3Bv2）的菌株在凉爽的热带高原和温带地区引起疾病。 R3Bv2尚未在北美建立，但由于担心会破坏美国马铃薯产业，因此将其指定为精选代理，并受到严格的检疫法规的约束。对R3Bv2进行隔离筛选需要适用于植物组织或土壤中存在的少量种群的快速且稳定的分析方法，并且必须将R3Bv2与多个其他的茄形青枯菌亚组区分开。我们开发了100％准确的实时聚合酶链反应（RT-PCR）分析法，可以检测R3Bv2群体> 1,000个细胞ml（-1）。但是，某些植物和土壤样品中存在的化合物抑制了RT-PCR的检测。因此，我们开发了简单的免疫磁分离（IMS）和磁捕获杂交（MCH）方法来从PCR抑制剂中纯化茄形红枯菌细胞或DNA。当与RT-PCR结合使用时，这些工具允许在直接RT-PCR失败的情况下在茎，块茎和土壤样品中以> 500细胞ml（-1）的水平检测R3Bv2，并将检测时间从数天缩短为数小时。 IMS-RT-PCR通常比MCH-RT-PCR更为敏感，尤其是在人群较低的情况下。

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