Improved detection methods for fruit tree phytoplasmas
Improved detection methods for fruit tree phytoplasmas
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DOI：
10.1007/bf02772160
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发表时间：
2001-06-01
影响因子：
2.1
通讯作者：
Machado, MLD
中科院分区：
文献类型：
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作者：
Heinrich, M;Botti, S;Machado, MLD
感染果树的植原体在欧洲和北美被认为是检疫性生物。由于它们在寄主植物中的分布极不规则，检测常常受到阻碍。敏感、特异和快速的诊断测试对于常规检测将是高度期望的，主要是为了避免使用受感染的种植材料。PCR方法需要繁琐的DNA制备：此外，可用的引物是高度特异性的，并表现出一些同源性叶绿体和质体DNA。为了解决这些问题，我们比较了几种DNA制备方案的DNA纯度，成本和所需的时间。我们还开发了新的引物使用rDNA序列信息从奥地利分离的欧洲核果黄（ESFY）。这些引物在高退火温度下操作，因此增加了特异性并降低了假阳性的风险。该引物可以可靠地检测欧洲植原体（AP，ESFY和PD）内保持在微繁殖长春花分离物的集合。因此，它们适合作为植原体检测的通用引物。引物也可用于菌株鉴定，通过直接PCR，然后进行RFLP分析，如用微繁殖果树材料证明的。最后，IC-PCR方法，使用引物检测AP被发现是非常敏感的，适用于大规模的测试苹果材料在体内和体外。
Phytoplasmas infecting fruit trees are considered quarantine organisms in Europe and North America. Detection often is hampered by their extremely irregular distribution in host plants. A sensitive, specific and quick diagnostic test would be highly desirable for routine detection, mainly to avoid using infected planting material. PCR methods require tedious preparation of DNA: also, the available primers are highly specific and exhibit some homology to chloroplast and plastid DNA. To address these problems, we compared several DNA preparation protocols for purity of DNA, cost and time required. We also developed new primers using rDNA sequence information from an Austrian isolate of European Stone Fruit Yellows (ESFY). These primers operate at high annealing temperatures and, thus, increase the specificity and decrease the risk of false positives. The primers could reliably detect the European phytoplasmas (AP, ESFY and PD) within a collection of isolates maintained in micropropagated periwinkle. Thus, they are suitable as general primers for phytoplasma detection. The primers also can be used for strain identification by direct PCR followed by RFLP analysis as demonstrated with micropropagated fruit tree material. Finally, an IC-PCR method that uses the primers for AP detection was found very sensitive and suitable for large-scale testing of apple material in vivo and in vitro.