Miles Timothy D, Martin Frank N, Robideau Gregg P, Bilodeau Guillaume J, Coffey Michael D
School of Natural Sciences, California State University Monterey Bay, Seaside, CA.
Crop Improvement and Protection Research Unit, USDA-ARS, Salinas, CA.
Plant Dis. 2017 Jul;101(7):1162-1170. doi: 10.1094/PDIS-09-16-1224-RE. Epub 2017 Apr 19.
The genus Phytophthora contains many invasive species to the U.S.A. that have the potential to cause significant damage to agriculture and native ecosystems. A genus and species-specific diagnostic assay was previously reported based on mitochondrial gene order differences that allowed for the systematic development of 14 species-specific TaqMan probes for pathogen detection ( Bilodeau et al. 2014 ). In this study, an additional 32 species-specific TaqMan probes for detection of primarily invasive species have been validated against 145 Phytophthora taxa as well as a range of Pythium and plant DNA samples. All validated probes were found to be species-specific and could be multiplexed with a genus-specific probe. The lower limit of linear detection using purified genomic DNA ranged from 1 to 100 fg in all assays. In addition, 124 unique TaqMan probes for Phytophthora spp. developed in silico are presented, which, if testing confirms they are species-specific, will provide diagnostic capabilities for approximately 89% of the genus. To enhance sensitivity of detection for several species that contained a single nucleotide polymorphism (SNP) in the reverse primer, a second primer was developed that is added in a small amount to the master mix. Furthermore, a PCR-RFLP system was developed that could be used to identify individual species when multiple species are present in a sample, without requiring cloning or sequencing. Several experiments were also conducted to compare various qPCR thermal cyclers and independent validation experiments with another research laboratory to identify possible limitations when the assays are used on a range of equipment in different labs. This system represents a comprehensive, hierarchal approach to increase the detection capability and provide tools to help prevent the introduction of invasive Phytophthora species.
疫霉属包含许多入侵美国的物种，这些物种有可能对农业和本地生态系统造成重大损害。此前曾报道过一种基于线粒体基因顺序差异的属和种特异性诊断检测方法，该方法允许系统开发14种用于病原体检测的种特异性TaqMan探针（比洛多等人，2014年）。在本研究中，另外32种用于检测主要入侵物种的种特异性TaqMan探针已针对145个疫霉分类群以及一系列腐霉属和植物DNA样本进行了验证。所有经过验证的探针均具有种特异性，并且可以与一种属特异性探针进行多重检测。在所有检测中，使用纯化基因组DNA的线性检测下限范围为1至100 fg。此外，还展示了通过计算机模拟开发的124种针对疫霉属物种的独特TaqMan探针，如果测试证实它们具有种特异性，将为该属约89%的物种提供诊断能力。为了提高对几种在反向引物中含有单核苷酸多态性（SNP）的物种的检测灵敏度，开发了第二种引物，并以少量添加到主混合物中。此外，还开发了一种PCR-RFLP系统，当样本中存在多个物种时，该系统可用于识别单个物种，而无需克隆或测序。还进行了几项实验，比较了各种qPCR热循环仪，并与另一个研究实验室进行了独立验证实验，以确定在不同实验室的一系列设备上使用这些检测方法时可能存在的局限性。该系统代表了一种全面、分层的方法，可提高检测能力，并提供工具以帮助防止入侵疫霉物种的引入。
