van Doorn Ronald, Szemes Marianna, Bonants Peter, Kowalchuk George A, Salles Joana F, Ortenberg Elen, Schoen Cor D
Plant Research International B,V,, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.
BMC Genomics. 2007 Aug 14;8:276. doi: 10.1186/1471-2164-8-276.
Diagnostics and disease-management strategies require technologies to enable the simultaneous detection and quantification of a wide range of pathogenic microorganisms. Most multiplex, quantitative detection methods available suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. Here, we demonstrate the efficacy of a novel, high-throughput, ligation-based assay for simultaneous quantitative detection of multiple plant pathogens. The ligation probes, designated Plant Research International-lock probes (PRI-lock probes), are long oligonucleotides with target complementary regions at their 5' and 3' ends. Upon perfect target hybridization, the PRI-lock probes are circularized via enzymatic ligation, subsequently serving as template for individual, standardized amplification via unique probe-specific primers. Adaptation to OpenArrays, which can accommodate up to 3072 33 nl PCR amplifications, allowed high-throughput real-time quantification. The assay combines the multiplex capabilities and specificity of ligation reactions with high-throughput real-time PCR in the OpenArray, resulting in a flexible, quantitative multiplex diagnostic system.
The performance of the PRI-lock detection system was demonstrated using 13 probes targeting several significant plant pathogens at different taxonomic levels. All probes specifically detected their corresponding targets and provided perfect discrimination against non-target organisms with very similar ligation target sites. The nucleic acid targets could be reliably quantified over 5 orders of magnitude with a dynamic detection range of more than 104. Pathogen quantification was equally robust in single target versus mixed target assays.
This novel assay enables very specific, high-throughput, quantitative detection of multiple pathogens over a wide range of target concentrations and should be easily adaptable for versatile diagnostic purposes.
诊断和疾病管理策略需要能够同时检测和定量多种致病微生物的技术。现有的大多数多重定量检测方法在多重检测水平、通量和定量准确性之间存在权衡。在此，我们展示了一种基于连接的新型高通量检测方法用于同时定量检测多种植物病原体的有效性。连接探针，称为国际植物研究锁定探针（PRI锁定探针），是长寡核苷酸，在其5'和3'末端具有靶标互补区域。在与靶标完美杂交后，PRI锁定探针通过酶促连接环化，随后作为通过独特的探针特异性引物进行个体标准化扩增的模板。适配可容纳多达3072个33 nl PCR扩增的OpenArrays，实现了高通量实时定量。该检测方法将连接反应的多重能力和特异性与OpenArray中的高通量实时PCR相结合，形成了一个灵活的定量多重诊断系统。
使用针对不同分类水平的几种重要植物病原体的13种探针展示了PRI锁定检测系统的性能。所有探针均能特异性检测其相应靶标，并能很好地区分具有非常相似连接靶标位点的非靶标生物体。核酸靶标在超过5个数量级的范围内能够可靠定量，动态检测范围超过104。在单靶标与混合靶标检测中，病原体定量同样可靠。
这种新型检测方法能够在很宽的靶标浓度范围内对多种病原体进行非常特异、高通量的定量检测，并且应该很容易适用于多种诊断目的。
