Low-High-Low Rotationally Pulse-Actuated Serial Dissolvable Film Valves Applied to Solid Phase Extraction and LAMP Isothermal Amplification for Plant Pathogen Detection on a Lab-on-a-Disc.

The ability of the centrifugal Lab-on-a-Disc (LoaD) platform to closely mimic the "on bench" liquid handling steps (laboratory unit operations (LUOs)) such as metering, mixing, and aliquoting supports on-disc automation of bioassay without the need for extensive biological optimization. Thus, well-established bioassays, normally conducted manually using pipettes or using liquid handling robots, can be relatively easily automated in self-contained microfluidic chips suitable for use in point-of-care or point-of-use settings. The LoaD's ease of automation is largely dependent on valves that can control liquid movement on the rotating disc. The optimum valving strategy for a true low-cost and portable device is rotationally actuated valves, which are actuated by changes in the disc spin-speed. However, due to tolerances in disc manufacturing and variations in reagent properties, most of these valving technologies have inherent variation in their actuation spin-speed. Most valves are actuated through stepped increases in disc spin-speed until the motor reaches its maximum speed (rarely more than 6000 rpm). These manufacturing tolerances combined with this "analogue" mechanism of valve actuation limits the number of LUOs that can be placed on-disc. In this work, we present a novel valving mechanism called low-high-low serial dissolvable film (DF) valves. In these valves, a DF membrane is placed in a dead-end pneumatic chamber. Below an actuation spin-speed, the trapped air prevents liquid wetting and dissolving the membrane. Above this spin-speed, the liquid will enter and wet the DF and open the valve. However, as DFs take ∼40 s to dissolve, the membrane can be wetted, and the disc spin-speed reduced before the film opens. Thus, by placing valves in a series, we can govern on which "digital pulse" in spin-speeding a reagent is released; a reservoir with one serial valve will open on the first pulse, a reservoir with two serial valves on the second, and so on. This "digital" flow control mechanism allows the automation of complex assays with high reliability. In this work, we first describe the operation of the valves, outline the theoretical basis for their operation, and support this analysis with an experiment. Next, we demonstrate how these valves can be used to automate the solid-phase extraction of DNA on on-disc LAMP amplification for applications in plant pathogen detection. The disc was successfully used to extract and detect, from a sample lysed off-disc, DNA indicating the presence of thermally inactivated Clavibacter michiganensis ssp. michiganensis (Cmm), a bacterial pathogen on tomato leaf samples.

Plant pathogen detection on a lab-on-a-disc using solid-phase extraction and isothermal nucleic acid amplification enabled by digital pulse-actuated dissolvable film valves.

By virtue of its ruggedness, portability, rapid processing times, and ease-of-use, academic and commercial interest in centrifugal microfluidic systems has soared over the last decade. A key advantage of the LoaD platform is the ability to automate laboratory unit operations (LUOs) (mixing, metering, washing etc.) to support direct translation of 'on-bench' assays to 'on-chip'. Additionally, the LoaD requires just a low-cost spindle motor rather than specialized and expensive microfluidic pumps. Furthermore, when flow control (valves) is implemented through purely rotational changes in this same spindle motor (rather than using additional support instrumentation), the LoaD offers the potential to be a truly portable, low-cost and accessible platform. Current rotationally controlled valves are typically opened by sequentially increasing the disc spin-rate to a specific opening frequency. However, due lack of manufacturing fidelity these specific opening frequencies are better described as spin frequency 'bands'. With low-cost motors typically having a maximum spin-rate of 6000 rpm (100 Hz), using this 'analogue' approach places a limitation on the number of valves, which can be serially actuated thus limiting the number of LUOs that can be automated. In this work, a novel flow control scheme is presented where the sequence of valve actuation is determined by architecture of the disc while its timing is governed by freely programmable 'digital' pulses in its spin profile. This paradigm shift to 'digital' flow control enables automation of multi-step assays with high reliability, with full temporal control, and with the number of LUOs theoretically only limited by available space on the disc. We first describe the operational principle of these valves followed by a demonstration of the capability of these valves to automate complex assays by screening tomato leaf samples against plant pathogens. Reagents and lysed sample are loaded on-disc and then, in a fully autonomous fashion using only spindle-motor control, the complete assay is automated. Amplification and fluorescent acquisition take place on a custom spin-stand enabling the generation of real-time LAMP amplification curves using custom software. To prevent environmental contamination, the entire discs are sealed from atmosphere following loading with internal venting channels permitting easy movement of liquids about the disc. The disc was successfully used to detect the presence of thermally inactivated Clavibacter michiganensis. Michiganensis (CMM) bacterial pathogen on tomato leaf samples.

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.

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.

Fungal Communities Including Plant Pathogens in Near Surface Air Are Similar across Northwestern Europe.

Information on the diversity of fungal spores in air is limited, and also the content of airborne spores of fungal plant pathogens is understudied. In the present study, a total of 152 air samples were taken from rooftops at urban settings in Slagelse, DK, Wageningen NL, and Rothamsted, UK together with 41 samples from above oilseed rape fields in Rothamsted. Samples were taken during 10-day periods in spring and autumn, each sample representing 1 day of sampling. The fungal content of samples was analyzed by metabarcoding of the fungal internal transcribed sequence 1 (ITS1) and by qPCR for specific fungi. The metabarcoding results demonstrated that season had significant effects on airborne fungal communities. In contrast, location did not have strong effects on the communities, even though locations were separated by up to 900 km. Also, a number of plant pathogens had strikingly similar patterns of abundance at the three locations. Rooftop samples were more diverse than samples taken above fields, probably reflecting greater mixing of air from a range of microenvironments for the rooftop sites. Pathogens that were known to be present in the crop were also found in air samples taken above the field. This paper is one of the first detailed studies of fungal composition in air with the focus on plant pathogens and shows that it is possible to detect a range of pathogens in rooftop air samplers using metabarcoding.

Tetra-primer ARMS PCR for rapid detection and characterisation of Plasmopara viticola phenotypes resistant to carboxylic acid amide fungicides.

BACKGROUND: The occurrence of Plasmopara viticola populations resistant to carboxylic acid amide (CAA) fungicides is becoming a serious problem in the control of grapevine downy mildew worldwide. The resistance is caused by point mutations in the PvCesA3 gene. These isolates with this mutation have been detected mainly by determining the minimum inhibitory concentration of fungicides, which is always time consuming and inefficient. RESULTS: To establish a suitable method for rapid detection of the G1105S mutation in P. viticola, an efficient and simple molecular method was developed, based on tetra-primer ARMS PCR. A set of four primers were designed and optimised to distinguish the different genotypes within one step. Only 2 h was required from the sampling of symptoms to the phenotyping of fungicide resistance. Using this method, CAA-resistant P. viticola were identified for the first time in China. Also, the finding of sensitive heterozygotes indicated that the resistant allele is spreading in the population in Ziyuan. CONCLUSION: This new method proved to be useful as an early warning system for resistance outbreaks of P. viticola to CAA fungicides in the field and may be helpful in decisions concerning rotation of different fungicide groups. © 2016 Society of Chemical Industry.

Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola.

A rapid LAMP (loop-mediated isothermal amplification) detection method was developed on the basis of the ITS sequence of P. viticola, the major causal agent of grape downy mildew. Among the 38 fungal and oomycete species tested, DNA isolated exclusively from P. viticola resulted in a specific product after LAMP amplification. This assay had high sensitivity and was able to detect the presence of less than 33 fg of genomic DNA per 25-µL reaction within 30 min. The infected leaves may produce sporangia that serve as a secondary inoculum. The developed LAMP assay is efficient for estimating the latent infection of grape leaves by P. viticola. When combined with the rapid and simple DNA extraction method, this assay's total detection time is shortened to approximately one hour; therefore it is suitable for on-site detection of latent infection in the field. The sporangia levels in the air are strongly associated with disease severity. The LAMP method was also demonstrated to be able to estimate the level of sporangia released in the air in a certain period. This assay should make disease forecasting more accurate and rapid and should be helpful in decision-making regarding the control of grape downy mildew.

A universal microarray detection method for identification of multiple Phytophthora spp. using padlock probes.

The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5' and 3' ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.

Molecular techniques for pathogen identification and fungus detection in the environment.

Many species of fungi can cause disease in plants, animals and humans. Accurate and robust detection and quantification of fungi is essential for diagnosis, modeling and surveillance. Also direct detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great many fungi are difficult or impossible to cultivate. In the last decade, effective amplification platforms, probe development and various quantitative PCR technologies have revolutionized research on fungal detection and identification. Examples of the latest technology in fungal detection and differentiation are discussed here.

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 undetected.

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

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. RESULTS: 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. CONCLUSION: 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.

Use of hybridization melting kinetics for detecting Phytophthora species using three-dimensional microarrays: demonstration of a novel concept for the differentiation of detection targets.

Microarray-based detection is limited by variable and inconsistent hybridization intensities across the diversity of probes used in each array. In this paper, we introduce a novel concept for the differentiation of detection targets using duplex melting kinetics. A microarray assay was developed on a PamChip microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridization intensity, and enabled the detection of individual Phytophthora species and mixtures thereof.

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.

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.