C.D. Schoen

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.

Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction.

BackgroundTo maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products.ResultsIn this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation.In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR.ConclusionCompared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetected.

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

ABSTRACT Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5′ and 3′ ends which confer extremely specific target detection. Probes also incorporate a desthiobiotin moiety and an internal endonuclease IV cleavage site. DNA samples are PCR amplified, and the resulting products serve as potential targets for PLP ligation. Upon perfect target hybridization, the PLPs are circularized via enzymatic ligation, captured, and cleaved, allowing only the originally ligated PLPs to be visualized on a universal microarray. Unlike previous procedures, the probes themselves are not amplified, thereby allowing a simple PLP cleavage to yield a background-free assay. We designed and tested nine PLPs targeting several oomycetes and fungi. All of the probes specifically detected their corresponding targets and provided perfect discrimination against closely related nontarget organisms, yielding an assay sensitivity of 1 pg genomic DNA and a dynamic detection range of 104. A practical demonstration with samples collected from horticultural water circulation systems was performed to test the robustness of the newly developed multiplex assay. This novel LD system enables highly specific detection and identification of multiple pathogens over a wide range of target concentrations and should be easily adaptable to a variety of applications in environmental microbiology.

Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays

ABSTRACT PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42:1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms.

Design of molecular beacons for AmpliDet RNA assay—Characterization of binding stability and probe specificity

AmpliDet RNA is a real-time diagnostic method, the specificity of which is defined mainly by the molecular beacon (MB). MBs can be characterized according to the stability of their stem-and-loop structures and that of the probe-target duplex via the free energies accompanying their formation. By the application of thermodynamic models, we propose a prediction method for these deltaG(0) parameters, which was compared to experimental analysis. The average absolute discrepancies for deltaG(0)(41) and for the melting temperatures of MB secondary structures were 0.30 +/- 0.26 kcal/mol and 2.15 +/- 1.5 degrees C, respectively. deltaG(0)(41) of probe-target interaction was predicted with a discrepancy of 1.2 +/- 1.0 kcal/mol. To characterize specificity, we formulated a model system with several MBs of highly similar sequence, but different lengths, and template RNAs carrying different types of mutations. We demonstrated the ability to detect a point mutation, or to tolerate one, irrespective of mismatch type. Of the nucleotide analogues tested, universal pyrimidine was found to increase MB tolerance substantially toward polymorphism. In the present study MBs were characterized under AmpliDet RNA conditions, with respect to probe stability, binding strength, and specificity, which led us to propose a design method, useful for probe design for AmpliDet RNA and adaptable to microarrays.

New Technologies for Sensitive and Specific Routine Detection of Plant Pathogenic Bacteria

In the last decade, nucleic acid sequence based amplification methods have been developed for many important plant pathogenic bacteria. Although they possess excellent properties with respect to sensitivity and specificity, the introduction of these techniques for routine detection has been hampered by in particular the lack of robustness. In different ring tests, both for human and plant pathogens, the failures of PCR amplification to diagnose correctly infected and non-infected sample material have been demonstrated many times. Carry-over contamination of amplicons has been recognized as the main source of false-positive results, whereas the presence of PCR inhibiting components in sample extracts is known as the main reason for false-negative results. In this paper new technologies and approaches are described to improve the quality of DNA and RNA amplification methods for routine detection.