Marzachí Cristina, Bosco Domenico
CNR, Istituto di Virologia Vegetale, Strada delle Cacce, 73, Torino, 10135, Italy.
Mol Biotechnol. 2005 Jun;30(2):117-28. doi: 10.1385/MB:30:2:117.
A real-time polymerase chain reaction (PCR) method for the quantification of chrysanthemum yellows (CY) phytoplasma DNA in its plant (Chrysanthemum carinatum) and insect (Macrosteles quadripunctulatus) host is described. The quantity of CY DNA was measured in each run relative to the amount of host DNA in the sample. Primers and a TaqMan probe for the specific PCR amplification of phytoplasma DNA were designed on a cloned CY-specific ribosomal fragment. Primers and TaqMan probes were also designed on sequences of the internal transcribed spacer region of the insect's ITS1 rDNA and of the plant's 18S rDNA for amplification from C. carinatum and M. quadripunculatus, respectively. Absolute quantification of CY DNA was achieved by comparison with a dilution series of the plasmid containing a CY 16S rDNA target sequence. Absolute quantification of plant and insect DNAs was achieved by comparison with a dilution series of the corresponding DNAs. Quantification of CY DNA in relation to host DNA was finally expressed as genome units (GU) of phytoplasma DNA per nanogram of host DNA. Relative quantification avoided influences due to different yields during the DNA extraction procedure. The quantity of CY DNA was about 10,000-20,000 GU/ng of plant DNA and about 30,000-50,000 GU/ng of insect DNA. The method described could be used to phytoplasma multiplication and movement in different plant and insect hosts.
本文描述了一种实时聚合酶链反应（PCR）方法，用于定量分析菊花黄化（CY）植原体DNA在其植物宿主（勋章菊）和昆虫宿主（四斑大叶蝉）中的含量。每次运行时，CY DNA的含量是相对于样品中宿主DNA的量进行测量的。用于植原体DNA特异性PCR扩增的引物和TaqMan探针是基于克隆的CY特异性核糖体片段设计的。还分别根据昆虫ITS1 rDNA的内部转录间隔区序列和植物18S rDNA的序列设计了引物和TaqMan探针，用于从勋章菊和四斑大叶蝉中进行扩增。通过与含有CY 16S rDNA靶序列的质粒稀释系列进行比较，实现了CY DNA的绝对定量。通过与相应DNA的稀释系列进行比较，实现了植物和昆虫DNA的绝对定量。CY DNA相对于宿主DNA的定量最终表示为每纳克宿主DNA中植原体DNA的基因组单位（GU）。相对定量避免了DNA提取过程中不同产量带来的影响。CY DNA的含量约为每纳克植物DNA 10,000 - 20,000 GU，每纳克昆虫DNA 30,000 - 50,000 GU。所描述的方法可用于研究植原体在不同植物和昆虫宿主中的增殖和移动情况。
