Hansen Z R, Knaus B J, Tabima J F, Press C M, Judelson H S, Grünwald N J, Smart C D
Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Geneva, NY, USA.
Horticultural Crops Research Laboratory, USDA Agricultural Research Service, Corvallis, OR, USA.
J Appl Microbiol. 2016 Apr;120(4):1010-20. doi: 10.1111/jam.13079. Epub 2016 Mar 7.
To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA.
Two sets of loop-mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross-reacted with the closely related species Phytophthora nicotianae. Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross-react with P. nicotianae. The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis and P. phaseoli. Cross-reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions.
Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity.
This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.
设计并验证一种用于快速检测致病疫霉DNA的比色环介导等温扩增检测方法。
设计了两组环介导等温扩增（LAMP）引物，并评估了它们对致病疫霉的敏感性和特异性。ITSII引物靶向核糖体DNA内部转录间隔区的一部分。这些引物对致病疫霉DNA的检测限为2 pg，且与近缘物种烟草疫霉发生交叉反应。为提高检测特异性而设计的Rgn86_2引物靶向一种保守假设蛋白的一部分。这些引物对致病疫霉DNA的检测限为200 pg，且不与烟草疫霉发生交叉反应。使用近缘物种安第斯致病疫霉、甘薯疫霉、奇异致病疫霉和菜豆致病疫霉进一步测试了Rgn86_2检测方法的特异性。与安第斯致病疫霉和奇异致病疫霉发生了交叉反应，但这两个物种均不侵染番茄或马铃薯。两组引物均能够检测从番茄晚疫病叶斑中提取的致病疫霉DNA。
两种比色LAMP检测方法均能从纯培养物以及受感染的叶片组织中检测到致病疫霉DNA。ITSII引物具有更高的敏感性，而Rgn86_2引物具有更高的特异性。
这是关于用于检测马铃薯和番茄晚疫病的致病因子致病疫霉的LAMP检测方法的首次报道。这些检测方法在植物病害研究和诊断实验室中具有直接应用的潜力。
