Osman Fatima, Rowhani Adib
Department of Plant Pathology, University of California, Davis, 95616, USA.
J Virol Methods. 2006 May;133(2):130-6. doi: 10.1016/j.jviromet.2005.11.005. Epub 2005 Dec 6.
An extraction technique for reverse transcription-PCR (RT-PCR) detection of plant pathogens including viruses, bacteria and phytoplasma is described. The total nucleic acid of these plant pathogens was obtained by direct spotting of crude sap derived from infected leaf, petiole or cambial tissue onto two different types of membranes, positively charged Hybond N(+) Nylon and FTA membranes, and processed for use in PCR. Thirteen different plant viruses, Xylella fastidiosa (causal agent of Pierce's disease) and phytoplasmas were included in the experiment. A thermal treatment (95 degrees C for 10 min) of the Hybond N(+) Nylon discs in a buffered solution improved the detection, but for FTA membrane discs the thermal treatment was not required and the discs were directly placed in the PCR reaction cocktail. Specific amplification of genomic or ribosomal RNA fragments of these pathogens was obtained by one-step RT-PCR except for X. fastidiosa in which a fragment of the genomic DNA was used for amplification. The same sample preparation methods also worked well for real-time RT-PCR (TaqMan). The sample preparation techniques reported here could be used to store samples for future PCR test or for long distance shipment to a detection laboratory.
本文描述了一种用于逆转录聚合酶链反应（RT-PCR）检测植物病原体（包括病毒、细菌和植原体）的提取技术。这些植物病原体的总核酸是通过将来自受感染叶片、叶柄或形成层组织的粗汁液直接点样到两种不同类型的膜上获得的，即带正电荷的Hybond N(+)尼龙膜和FTA膜，然后进行处理以用于PCR。实验中包括了13种不同的植物病毒、桑萎蔫病菌（皮尔斯病的病原体）和植原体。在缓冲溶液中对Hybond N(+)尼龙膜片进行热处理（95℃，10分钟）可提高检测效果，但对于FTA膜片则无需热处理，可直接将膜片放入PCR反应混合液中。除了桑萎蔫病菌使用基因组DNA片段进行扩增外，通过一步RT-PCR可对这些病原体的基因组或核糖体RNA片段进行特异性扩增。相同的样品制备方法对实时RT-PCR（TaqMan）也同样适用。本文报道的样品制备技术可用于储存样品以便未来进行PCR检测，或用于长途运输至检测实验室。
