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

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

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

Background To 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. Results In 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. Conclusion Compared 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 undetecte

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

Calibration curves to assess the reproducibility of the Biotrove OpenArray™ platform and the PRI-lock system

<p><b>Copyright information:</b></p><p>Taken from "Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™"</p><p>http://www.biomedcentral.com/1471-2164/8/276</p><p>BMC Genomics 2007;8():276-276.</p><p>Published online 14 Aug 2007</p><p>PMCID:PMC2064939.</p><p></p> () Inter-array variation: The PRI-lock probe mixture was ligated on a 10-fold serial dilution of target and amplified on the Biotrove OpenArray™ platform. Samples were tested on three different OpenArrays™ (n = 3). Data represent averages of six PCR replicates (n = 6). The error bars represent the standard deviations. () Assay-to-assay reproducibility: Three separate whole assay repeats ligation reactions were performed on 10-fold serial dilution of target and amplified in the Biotrove OpenArray™ platform. Data represent averages of 3 separate ligation experiments (n = 3) with each, four PCR replicates (n = 4). Cvalues were normalized using the ILC PRI-lock probe. The error bars represent the standard deviations (Sometimes the standard deviation error bars fall within the data squares)

SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3′ end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5′ end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.BN/Stan Brouns La