Highly scalable combinatorial mixing of samples with target-specific primers for rapid pathogen detection on a centrifugal platform

In application areas such as crop genotyping, plant diagnostics, pharmaceuticals and forensics, screening a large number of M samples for specific responses to a library of N active agents in a time- and cost-efficient manner is of critical importance. Parameters of interest include response of cells to a specific drug compound, identification of specific genes or plant pathogens in crops using DNA markers and DNA traceability for food safety. The cost of reagents as well as the liquid handling ro-botics required to perform the enormous number of pipetting steps severely hamper the proliferation of such key technologies into smaller laboratories

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

Patient safety culture measurement in general practice. Clinimetric properties of 'SCOPE'

<p>Abstract</p> <p>Background</p> <p>A supportive patient safety culture is considered to be an essential condition for improving patient safety. Assessing the current safety culture in general practice may be a first step to target improvements. To that end, we studied internal consistency and construct validity of a safety culture questionnaire for general practice (SCOPE) which was derived from a comparable questionnaire for hospitals (Dutch-HSOPS).</p> <p>Methods</p> <p>The survey was conducted among caregivers of Dutch general practice as part of an ongoing quality accreditation process using a 46 item questionnaire. We conducted factor analyses and studied validity by calculating correlations between the subscales and testing the hypothesis that respondents' <it>patient safety grade </it>of their practices correlated with their scores on the questionnaire.</p> <p>Results</p> <p>Of 72 practices 294 respondents completed the questionnaire. Eight factors were identified concerning <it>handover and teamwork, support and fellowship, communication openness, feedback and learning from error, intention to report events, adequate procedures and staffing, overall perceptions of patient safety </it>and <it>expectations and actions of managers</it>. Cronbach's alpha of the factors rated between 0.64 and 0.85. The subscales intercorrelated moderately, except for the factor about intention to report events. Respondents who graded patient safety highly scored significantly higher on the questionnaire than those who did not.</p> <p>Conclusions</p> <p>The SCOPE questionnaire seems an appropriate instrument to assess patient safety culture in general practice. The clinimetric properties of the SCOPE are promising, but future research should confirm the factor structure and construct of the SCOPE and delineate its responsiveness to changes in safety culture over time.</p

Loop-mediated isothermal amplification (LAMP) shield for Arduino DNA detection

Objective: Loop-mediated isothermal amplification (LAMP) of DNA is gaining relevance as a method to detect nucleic acids, as it is easier, faster, and more powerful than conventional Polymerase Chain Reaction. However, LAMP is still mostly used in laboratory settings, because of the lack of a cheap and easy, one-button device that can perform LAMP experiments. Results: Here we show how to build and program an Arduino shield for a LAMP and detection of DNA. The here described Arduino Shield is cheap, easy to assemble, to program and use, it is battery operated and the detection of DNA is done by naked-eye so that it can be used in field

MOESM1 of Loop-mediated isothermal amplification (LAMP) shield for Arduino DNA detection

Additional file 1. Arduino LAMP shield electronics (Schematics and electronics used for the fabrication of the shield). Breakout of the prices (prices of single components). Fabrication of the heating block (Heating block fabrication using PDMS). Temperature test (temperature test of the heating block). Melting curve of the amplified gBlock (melting curve of the amplified product). Arduino LAMP shield source code (source code for the Arduino LAMP shield)

Automated DNA purification and Multiplexed lamp assay preparation On a centrifugal microfluidic “lab-on-a-disc” platform

This work presents a rotational-pulse actuated microfluidic cartridge enabling automated detection of plant pathogens on a compact device towards point-of-use monitoring of food safety. This highly integrated “Lab-on-a-Disc” (LoaD) system first runs the sample over a stationary phase of silica beads, followed by ethanol (EtOH) wash and final elution of DNA. The eluate is then homogenized using ‘shake mode’ agitation, accurately metered and then mixed with reagents for loop-mediated isothermal amplification (LAMP). We successfully purify plant DNA and demonstrate on-disc quantitative LAMP amplification