Miles Timothy D, Martin Frank N, Coffey Michael D
Phytopathology. 2015 Feb;105(2):265-78. doi: 10.1094/PHYTO-05-14-0134-R.
Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing, thereby reducing the time necessary for accurate diagnostics and making management decisions.
最近开发了几种等温扩增技术，这些技术能够耐受许多植物提取物中存在的抑制剂，从而减少了为进行诊断检测而获取纯化DNA的需求。一种与实时聚合酶链反应（PCR）在引物设计和标记探针方面有相似之处的市售技术是重组酶聚合酶扩增（RPA）。该技术被用于开发两种简单快速的检测疫霉属物种的方法：一种是与植物内部对照多重检测的属特异性检测方法，另一种是针对樟疫霉和克氏疫霉的种特异性检测方法。使用从136多个疫霉分类群、21个腐霉属物种、1个 Phytopythium 物种以及多种植物物种中提取的DNA，对所有检测方法的灵敏度（DNA范围从3 ng到1 fg）和特异性进行了测试。在所有病原体RPA检测中，使用纯化DNA的线性检测下限为200至300 fg DNA。测试了六种不同的提取缓冲液用于植物组织浸渍过程，并且通过从50多个不同宿主收集222个有症状的植物样本在田间对检测方法进行了验证。只有56个样本经培养检测疫霉属物种呈阳性，而使用疫霉属特异性RPA检测和TaqMan实时PCR检测时，有91个样本呈阳性。还开发了一种从阳性RPA扩增产物生成测序模板以确认物种鉴定的技术。这些RPA检测方法比传统技术具有更多优势，因为它们快速（最短15分钟即可获得结果），不需要DNA提取或大量培训即可完成，使用比基于PCR的检测方法更便宜的便携式设备，并且比当前基于免疫的方法特异性显著更高。这应该为病原体检测提供一种快速、可在现场部署的能力，将有助于样本采集点的处理，从而减少准确诊断和做出管理决策所需的时间。
