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

Abstract

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.

Oligonucleotide microarray methodology for taxonomic and functional monitoringof microbial community composition

“…a promising alternative for parallel quantification of microorganisms in environmental samples is based on the use of padlock probes (van Doorn et al 2007). With this approach, quantity is not inferred from hybridization signal but from taqMan real-time amplification of ligated probes.…”

Development and Experimental Validation of a Predictive Threshold Cycle Equation for Quantification of Virulence and Marker Genes by High-Throughput Nanoliter-Volume PCR on the OpenArray Platform

“…Low-volume PCR has been optimized in nanoliter-volume reactions by adjusting surface chemistry (33), ramping rates and decreased annealing temperatures (46), and adjusting the PCR master mix composition with extra Sybr green I, bovine serum albumin, and formamide added to the standard PCR mixture. Cross-platform comparisons between the PCR performed with the microplate format (10-and 20-l-volume reactions in the 7900HT) and with the OpenArray platform have shown high similarities in PCR efficiencies and detection limits (E. Ortenberg and D. Roberts, unpublished data; 7).…”

“…When many pathogens must be screened in parallel with the ability to characterize the uneven distribution of the associated VMGs and their allelic variability, optimization is necessary for high-throughput tools with high sensitivity and specificity, e.g., quantitative PCR (QPCR) (14,19,27,32,44,46,50,51). This optimization may be cumbersome because it requires the generation of multiple standard curves with the caveat that all primer sets must perform under the same amplification conditions.…”

“…One nonspecific primer targeting C. parvum was removed from the set due to false-positive observations. Thus, a total of 220 primer sets (109 used to establish the predictive C T (33,46). Two to four subarrays (each with 64 wells for 56 separate assays and eight loading controls) were used for each PCR sample.…”

Robust Detection and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay

“…PLPs are long oligonucleotides, ϳ100 bases long, containing target complementary arms at both termini of the probe. In the assay developed in this study, the target complementary arms are connected via a compound linker sequence containing spacer sequences, a thymine-linked desthiobiotin moiety for specific capture and release (25,53), deoxyuracil nucleotides for probe cleavage, and a unique sequence identifier, the so-called ZipCode, for standardized microarray hybridization (18) (Fig. 1A).…”