Licciardello G, Grasso F M, Bella P, Cirvilleri G, Grimaldi V, Catara V
Parco Scientifico e Tecnologico della Sicilia, Z. I. Blocco Palma I, Catania, Italy.
Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi di Catania, Catania, Italy.
Plant Dis. 2006 Dec;90(12):1523-1530. doi: 10.1094/PD-90-1523.
Phoma tracheiphila is the causal agent of a tracheomycotic disease of citrus called mal secco causing the dieback of twigs and branches. This pathogen is of quarantine concern; therefore, fast and reliable protocols are required to detect it promptly. A specific primer pair and a dual-labeled fluorogenic probe were used in a real-time polymerase chain reaction (PCR) with the Cepheid Smart Cycler II System (Transportable Device TD configuration) to detect this fungus in citrus samples. Real-time PCR assay was compared to modified conventional PCR assay. The sensitivity of the former was evaluated by testing P. tracheiphila DNA dilutions, and the minimum amount detectable was about 500 fg, whereas the linear quantification range was within 100 ng to 1 pg. Conventional PCR sensitivity was 10 pg. Conventional and real-time PCR successfully detected the fungus in woody samples of naturally infected lemon and artificially inoculated sour orange seedlings. Nevertheless, real-time PCR was about 10- to 20-fold more sensitive than conventional PCR, and preliminary results indicate that the former technique achieves quantitative monitoring of the fungus in tissues. Simple and rapid procedures to obtain suitable DNA samples from fungal cultures and citrus woody samples for PCR assays enable diagnosis to be completed in a short time.
柑桔茎点霉是一种导致柑桔发生干腐病的气管真菌病害的病原体，会致使嫩枝和树枝枯死。这种病原菌受到检疫关注；因此，需要快速且可靠的检测方案以便及时检测到它。在使用 Cepheid Smart Cycler II 系统（便携式设备 TD 配置）进行的实时聚合酶链反应（PCR）中，采用了一对特异性引物和一个双标记荧光探针来检测柑桔样品中的这种真菌。将实时 PCR 检测法与改良的常规 PCR 检测法进行了比较。通过检测柑桔茎点霉 DNA 稀释液评估了前者的灵敏度，可检测到的最小量约为 500 fg，而线性定量范围在 100 ng 至 1 pg 之间。常规 PCR 的灵敏度为 10 pg。常规 PCR 和实时 PCR 均成功检测到了自然感染柠檬的木质样品以及人工接种酸橙幼苗中的真菌。然而，实时 PCR 的灵敏度比常规 PCR 高约 10 至 20 倍，初步结果表明前一种技术能够对组织中的真菌进行定量监测。从真菌培养物和柑桔木质样品中获取适合 PCR 检测的 DNA 样品的简单快速程序，能够在短时间内完成诊断。
