Torres Ester, Bertolini Edson, Cambra Mariano, Montón Carmina, Martín María P
Laboratori Sanitat Vegetal, Generalitat de Catalunya, Via Circulació Nord Tram 6, 08040 Barcelona, Spain.
Mol Cell Probes. 2005 Oct;19(5):334-40. doi: 10.1016/j.mcp.2005.06.002.
A real time PCR assay conjugated with the fluorescent SYBR Green I dye has been developed for rapid, sensitive and quantitative detection of 'Ca. Phytoplasma pyri', 'Ca. P. prunorum' and 'Ca. P. mali', quarantine members of apple proliferation (16 SrX) group. The selected primers amplify specifically a target of 217-bp fragment from the 16 Sr gene region of the 16 SrX group and not from any other tested phytoplasma groups. An artificial template consisting in a plasmid clone of a 1785-bp DNA fragment of the 16S rRNA gene, 16S/23S rDNA spacer region, tRNA-Ile and partial 23S rRNA gene of a 'Ca. P. prunorum' isolate, was used to establish a calibration curve to evaluate the number of amplified targets per sample. The sensitivity of the technique was similar to nested-PCR (10 copies of the amplified target per microl). The estimated concentration of phytoplasmas in infected pear, plum and apricot trees ranged from 9.7 x 10(3) to 3.0 x 10(5) phytoplasmas per gram of tissue. The method offers the possibility to detect simultaneously, in a single reaction, all quarantine phytoplasmas affecting fruit trees hosts in Europe.
已开发出一种与荧光SYBR Green I染料结合的实时PCR检测方法，用于快速、灵敏和定量检测苹果增殖（16SrX）组的检疫性成员——梨植原体、李属植原体和苹果植原体。所选引物从16SrX组的16Sr基因区域特异性扩增出一个217bp片段的靶标，而不从任何其他测试的植原体组中扩增。使用一个人工模板，该模板由一个“李属植原体”分离株的16S rRNA基因、16S/23S rDNA间隔区、tRNA-Ile和部分23S rRNA基因的1785bp DNA片段的质粒克隆组成，用于建立校准曲线以评估每个样品中扩增靶标的数量。该技术的灵敏度与巢式PCR相似（每微升10个扩增靶标拷贝）。感染梨树、李树和杏树中植原体的估计浓度范围为每克组织9.7×10³至3.0×10⁵个植原体。该方法提供了在单一反应中同时检测影响欧洲果树寄主的所有检疫性植原体的可能性。
