Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology

Detection of genomic DNAs corresponding to individual (A–G) and complex pathogen samples (H–I) on a universal microarray

<p><b>Copyright information:</b></p><p>Taken from "Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays"</p><p>Nucleic Acids Research 2005;33(8):e70-e70.</p><p>Published online 28 Apr 2005</p><p>PMCID:PMC1087788.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The analysed targets were as follows: () , 1 ng; () , 1 ng; () , 1 ng; () AG 4-2, 1 ng; () , 1 ng; () , 1 ng; () , 1 ng; () , 500 pg; , 500 pg and , 500 pg; () , 500 pg; AG 4-2, 500 pg and , 500 pg; () , 500 pg; AG 4-1, 500 pg and , 500 pg; () , 0.5 pg and , 500 pg; and () , 500 pg and , 5 pg

Development of a Multiplexed Bead-Based Suspension Array for the Detection and Discrimination of Pospiviroid Plant Pathogens

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies

Details of pospiviroid isolates used for validation of the Luminex MagPlex-TAG pospiviroid array.

<p>Details of pospiviroid isolates used for validation of the Luminex MagPlex-TAG pospiviroid array.</p

Percentages of false positive and false negative results for Luminex MagPlex-TAG pospiviroid array data when analyzed using three different methods for setting thresholds for positivity.

<p>Percentages of false positive and false negative results for Luminex MagPlex-TAG pospiviroid array data when analyzed using three different methods for setting thresholds for positivity.</p

Predictions of the number of isolates of each pospiviroid species that will be detected using universal and species-specific assays of the Luminex MagPlex-TAG pospiviroid array.

<p>Predictions of the number of isolates of each pospiviroid species that will be detected using universal and species-specific assays of the Luminex MagPlex-TAG pospiviroid array.</p

Reproducibility of the Luminex MagPlex-TAG pospiviroid array over ten independent tests.

<p>One sample of <i>Potato spindle tuber viroid</i> (isolate #3077695) was tested in ten independent reactions over several days. Mean natural log (ln) median fluorescence intensity (MFI) values are plotted; error bars show plus or minus (±) one standard deviation. The horizontal bars plotted on each of the bars shows the detection threshold obtained from our kernel density estimation method, for each assay of the array. The small standard deviation of the ln(MFI) values for positively reacting assays in the array (PSTVd, PospUni and PlantIC) demonstrate the high level of precision of the multiplexed bead-based array.</p

Specificity of the Luminex MagPlex-TAG pospiviroid array when screened against sequence-characterized pospiviroid isolates.

<p>Performance of the 11-plex bead-based array when screened against a large panel of single and mixed infections of sequence-characterized pospiviroid isolates, obtained from natural infections of a variety of host plants (with mixed infections simulated). Data for healthy tomato (uninfected control) and blank (no template control) samples are included for reference. Asterisks denote mean natural log (ln) median fluorescence intensity (MFI) values that exceed the threshold for that assay within the multiplexed bead-based array.</p