Improved sampling methods for real-time polymerase chain reaction diagnosis of citrus canker from field samples
Improved sampling methods for real-time polymerase chain reaction diagnosis of citrus canker from field samples
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DOI：
10.1094/phyto.2004.94.1.61
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发表时间：
2004-01-01
期刊：
影响因子：
3.2
通讯作者：
Gabriel, DW
中科院分区：
文献类型：
--
作者：
Mavrodieva, V;Levy, L;Gabriel, DW
在过去的15年里，柑橘细菌性溃疡病至少三次被引入佛罗里达，尽管联邦和州进行了检疫和根除工作，但它仍在佛罗里达继续蔓延。准确、快速和可靠地检测致病因子是真实的重要的。然而，柑橘细菌性溃疡病是由至少两组遗传学上不同的柑橘黄单胞菌菌株引起的，并且在两组内存在寄主范围的差异。我们开发了一个最后的，敏感和可靠的实时聚合酶链反应（PCR）检测使用便携式，现场加固RAPID机器和引物设计检测所有溃疡致病菌株。开发了单病变取样方法，其需要最少的处理，并且允许在4 It的总时间内完成实时PCR诊断，并且具有来自患病病变的靶细胞的小于10 CFU的表观灵敏度。这种灵敏度允许首次对X进行分子检测。1912年溃疡病爆发前的植物标本馆里的柑橘。通过使用CaCO 3和Silwet L-77，以及通过最小化柑橘病变组织取样量或通过浸泡或擦拭但不研磨病变，显著提高了灵敏度。引物设计在特异性和灵敏度方面也具有重要意义。
Citrus bacterial canker disease has been introduced at least three times into Florida in the last 15 years and, despite federal and state quarantine and eradication efforts, continues to spread in Florida. Accurate, fast, and reliable detection of the causal agent is of,real importance. However, citrus bacterial canker is caused by at least two groups of phylogenetically distinct Xanthomonas citri strains, and there is host range variation within both groups. We developed a last, sensitive and reliable real-time polymerase chain reaction (PCR) assay using a portable, field-hardened RAPID machine and primers designed to detect all canker-causing strains. Single-lesion sampling methods were developed that required minimal handling and allowed complete real-time PCR diagnosis in a total time of 4 It and with an apparent sensitivity of less than 10 CFU of target cells from diseased lesions. This sensitivity allowed molecular detection for the first time of X. citri in a herbarium sample front a 1912 canker outbreak. Sensitivity was improved significantly by the use of CaCO3 and Silwet L-77, and by either minimizing the amount of citrus lesion tissue sampled or by soaking or swiping but not grinding the lesions. Primer design also was of significant importance in both specificity and sensitivity.