Aldaghi M, Massart S, Dutrecq O, Bertaccini A, Jijakli M H, Lepoivre P
Plant Pathology Unit, Gembloux Agricultural University (FUSAGx), Passage des déportés 2, 5030 Gembloux, Belgium.
J Virol Methods. 2009 Mar;156(1-2):96-101. doi: 10.1016/j.jviromet.2008.10.011. Epub 2008 Dec 18.
Different PCR protocols have been established for detection of European fruit trees phytoplasmas; however the majority of the procedures for extracting phytoplasma DNA are complex, time consuming, and expensive, with a risk of contamination or loss of target DNA. In present study, a crude extract preparation method previously used to detect other plant pathogens was adapted to samples from apple trees infected by 'Candidatus Phytoplasma mali'. End-point and real-time PCR detection of 'Ca. P. mali' were used to compare this extraction procedure with an established method for efficient extraction of purified DNA. The crude extract proved fully adequate for phytoplasma detection in samples from 86 in vitro and 35 in vivo apple shoots or plants and 10 periwinkle plants. High inter- and intra-run reproducibility was obtained for phytoplasma detection with different TaqMan MGB- or SYBR Green-based real-time PCR protocols applied to the crude extracts. Real-time PCR applied to serially diluted crude and purified extracts revealed the same phytoplasma detection limit (dilution up to 10(5)). All results confirm the suitability of this simple, quick, efficient extraction technique for accurate detection of 'Ca. P. mali' in different types of apple and periwinkle samples.
已经建立了不同的PCR方案用于检测欧洲果树林植原体；然而，大多数提取植原体DNA的程序复杂、耗时且昂贵，存在目标DNA污染或丢失的风险。在本研究中，一种先前用于检测其他植物病原体的粗提物制备方法被应用于感染‘苹果植原体’的苹果树样本。采用终点PCR和实时PCR检测‘苹果植原体’，将这种提取程序与一种已确立的高效提取纯化DNA的方法进行比较。结果表明，粗提物完全适用于检测86个离体和35个活体苹果嫩枝或植株样本以及10个长春花植株样本中的植原体。使用不同的基于TaqMan MGB或SYBR Green的实时PCR方案对粗提物进行植原体检测，获得了较高的批间和批内重现性。对系列稀释的粗提物和纯化提取物进行实时PCR检测，结果显示植原体检测限相同（稀释至10(5)）。所有结果证实了这种简单、快速、高效的提取技术适用于准确检测不同类型苹果和长春花样本中的‘苹果植原体’。
