Jawhari Maan, Abrahamian Peter, Sater Ali Abdel, Sobh Hana, Tawidian Patil, Abou-Jawdah Yusuf
Department of Agriculture, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut 1107 2020, Lebanon.
Department of Agriculture, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut 1107 2020, Lebanon.
Mol Cell Probes. 2015 Feb;29(1):63-70. doi: 10.1016/j.mcp.2014.12.003. Epub 2014 Dec 25.
Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.
杏仁丛枝病（AlmWB）是一种与“‘Ca. Phytoplasma phoenicium’”相关的、快速传播的杏仁、桃和油桃致死性病害。开发用于灵敏且特异检测植原体的PCR和定量实时PCR（qPCR）检测方法对于早期检测“Ca. P. phoenicium”以及进行流行病学研究至关重要。本文开发的qPCR检测方法使用了标记有Black Hole Quencher Plus的TaqMan®探针。PCR和qPCR检测方案的特异性在属于11个植原体16S rRNA组的17种植原体分离株上，以及在感染或未感染该植原体的杏仁、桃、油桃、本地植物和昆虫样本上进行了测试。所开发的检测方法对“Ca. P. phoenicium”显示出高特异性，对任何其他测试的植原体、植物或昆虫均无交叉反应。所开发的PCR和qPCR检测方法的灵敏度与使用通用引物的传统巢式PCR方案相似。通过对不同宿主、植物部位和潜在昆虫载体中的AlmWB植原体进行定量，进一步验证了qPCR检测方法。在杏仁树和油桃树茎和根的韧皮部组织中检测到“Ca. P. phoenicium”的最高滴度，每纳克宿主DNA中平均为10⁵至10⁶个基因组单位（GU/ng DNA）。新开发的PCR和qPCR方案是可靠、特异且灵敏的方法，易于应用于植物和昆虫中AlmWB的高通量诊断，可用于潜在载体和替代宿主的调查。
