Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
van Doorn
Ronald
R
Plant Research International B,V,, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands. ronald.vandoorn@wur.nl
Szemes
Marianna
M
Bonants
Peter
P
Kowalchuk
George A
GA
Salles
Joana F
JF
Ortenberg
Elen
E
Schoen
Cor D
CD
D001483:Base Sequence; D017931:DNA Primers; D015345:Oligonucleotide Probes; D010944:Plants / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D015345:Oligonucleotide Probes
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