Michael Glenn Mason

Amplification-based diagnostics has been widely used in plant disease diagnostics due to its sensitivity, rapidness, and flexibility. The modules to interpret amplification results rapidly, reliably, and easily in resource-limited settings (e.g., field-based environments) are in high demand. Therefore, we have designed and developed a simple, low-cost, easy-to-perform, and portable device named Dr. Diagnose to aid accurate, unbiased, and user-friendly readout of amplification reactions outside the model laboratory environment. Dr. Diagnose is an Arduino microcontroller-based light meter that can operate with a portable power bank. It allows the user to select to analyze either fluorescence or turbidity levels in amplification reactions by simply touching the button and informs the user of whether the reaction’s fluorescence or turbidity exceeds a set threshold and is thus considered positive within seconds. The sensitivity of Dr. Diagnose was found to be comparable to gel electrophoresis-based detection. Dr. Diagnose has a simple circuit containing only 13 electronic components, which means it can be easy to generate, affordable, and accessible to the broader community. The development of Dr. Diagnose provides a practical and inexpensive readout tool for amplifications at point-of-need in a rapid, accurate, and user-friendly manner. IMPORTANCE: The first critical step in timely disease management is rapid disease identification, which is ideally on-site detection. Of all the technologies available for disease identification, nucleic acid amplification-based diagnostics are often used due to their specificity, sensitivity, adaptability, and speed. However, the modules to interpret amplification results rapidly, reliably, and easily in resource-limited settings at point-of-need (PON) are in high demand. Therefore, we developed a portable, low-cost, and easy-to-perform device that can be used for amplification readout at PON to enable rapid yet reliable disease identification by users with minimal training.

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.

A number of isothermal DNA amplification technologies claim to be ideal for point-of-need (PON) applications as they enable reactions to be performed using a single-temperature heat source (e.g. water bath). Thus, we examined several isothermal amplification methods focusing on simplicity, cost, sensitivity and reproducibility to identify the most suitable method(s) for low resource PON applications. A number of methods were found unsuitable as they either involved multiple temperature incubations, were relatively expensive or required relatively large amounts target DNA for amplification. Among the methods examined, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) were found to be the most suitable for PON applications as they are both single step methods that provide highly sensitive and reproducible amplifications. The speed of LAMP reactions was greatly enhanced, up to 76%, with the addition of loop primers while the presence of swarm primers and the sequestration of free magnesium ions with nucleotides also enhanced the amplification speed. In contrast, we were unable to enhance RPA’s performance from the original published literature. While both RPA and LAMP have some drawbacks, either isothermal technology can reliably be used for on-site diagnostics with minimal equipment.

Food-borne diseases are a leading cause of illness and death in many developing countries and thus, there is a real need to develop affordable and practical technologies that can help improve food safety in these countries. The ability to efficiently identify food pathogens is essential to allow national regulatory authorities to monitor food quality and implement safety protocols. In this study, we have developed a simple, low-cost ($0.76 (USD)) complete food pathogen diagnostic workflow ideally suited for deployment in low-resource environments that uses a simple four step process (sample enrichment, cell lysis, DNA amplification, and naked-eye readout). The minimal number of steps and equipment involved in our diagnostic workflow, as well as the simplicity of the yes/no flocculation readout, allows non-technical personnel to perform and interpret the assay. To evaluate the system’s performance, we tested the entire system on fresh produce samples collected from local farms and markets in Cambodia for the presence of the E. coli O157 O-antigen polymerase, wzy . Although this was a proof-of-concept study, our system successfully revealed a clear correlation between the origin and condition of the produce collected and their likelihood of contamination. In conclusion, we believe that our easy-to-perform diagnostic system can have a significant impact on improving food quality and human health if adopted by regulatory authorities in developing countries due to the assay’s simplicity, affordability, and adaptability.

The plant hormone strigolactone (SL) inhibits shoot branching by suppressing the growth of axillary buds. This is thought to occur largely via regulation of the transcription factor BRANCHED1 (BRC1). Here, we clarify the central role of BRC1 and identify additional transcriptional responses by which SL might regulate axillary bud outgrowth in garden pea ( Pisum sativum ). We used a transcriptomic approach to identify differentially expressed transcripts in pea axillary buds in response to a synthetic SL, rac -GR24. Changes in transcript abundance were confirmed by measuring their response to GR24 ^5DS . BRC1 was required for the regulation of over half of the fourteen GR24 ^5DS -regulated genes, confirming its role as a mediator of SL transcriptional responses in axillary buds. All, but one, of the BRC1-dependent GR24 ^5DS -regulated genes were also regulated by branch-promoting treatments cytokinin (CK) and/or decapitation in an opposing manner to SL. This suggests that SL, CK, and decapitation regulate shoot branching via a common pathway. We used correlational analyses of gene co-expression data to infer a gene regulatory network consisting of nine key co-expression modules correlated with rac -GR24 treatment. Enrichment of GO terms such as cell proliferation, carbohydrate responses, and abscisic acid and jasmonic acid hormone pathways suggest a role for these in SL-mediated inhibition of shoot branching. In summary, we have shown that BRC1 is indeed a key transcriptional regulator of the SL signalling pathway in pea buds as well as a focal point of the SL, CK and decapitation signalling pathways to coordinate shoot branching in pea buds. One Sentence Summary: Identification of genes that are strigolactone-responsive and BRC1-dependent in pea buds reveals a high degree of overlap among strigolactone, cytokinin and decapitation response pathways.

Aims Current culture‐based methods for detection and determination of Campylobacter levels on processed chickens takes at least two days. Here, we sought to develop a new complete, low‐cost and rapid (approximately 2·5 hours) detection system requiring minimal operator input. Methods and Results We observed a strong correlation between culture‐based cell counts and our ability to detect either Campylobacter jejuni or Campylobacter coli by Loop mediated isothermal amplification (LAMP) from the same samples. This knowledge was used to develop a rapid and simple five‐step assay to quantify Campylobacter, which was subsequently assessed for its specificity, reproducibility and accuracy in quantifying Campylobacter levels from processed chickens. The assay was found to be highly specific for C. jejuni and C. coli and was capable of distinguishing between samples that are either within or exceeding the industry set target of 6,000 Campylobacter colony forming units (cfu) per carcass (equivalent to 12 cfu ml^‐1 of chicken rinse) with> 90% accuracy relative to culture‐based methods. Conclusions Our method can reliably quantify Campylobacter counts of processed chickens with an accuracy comparable to culture‐based assays but provides results within hours as opposed to days. Significance and Impact of the Study The research presented here will help improve food safety by providing fast Campylobacter detection that will enable the implementation of real‐time risk management strategies in poultry processing plants to rapidly test processed chickens and identify effective intervention strategies. This technology is a powerful tool that can be easily adapted for other organisms and thus could be highly beneficial for a broad range of industries.

A simple and highly reliable DNA amplification readout system for naked eye detection of amplified DNA in under 30 seconds.

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries. Nucleic acid amplification has proven to be indispensable in laboratories around the world for a myriad of applications from diagnostics to genotyping. The first step in any application aiming to amplify DNA or RNA is the extraction of nucleic acids from a complex biological sample; a task traditionally requiring specialised equipment, trained technicians, and multiple liquid handling steps. It is this complexity of current nucleic acid isolation methods that limit the use of many DNA amplification technologies outside of the modern laboratory environment. Therefore, in this study, we investigated new materials and approaches to simplify nucleic acid extraction. We found that cellulose-based filter paper can be used to rapidly bind nucleic acids, retain them during a short washing step to remove contaminants, and then elute them directly into the amplification reaction. We then adapted the cellulose filter to create a dipstick that can be used to purify nucleic acids from a wide range of plant, animal, and microbe samples in less than 30 seconds without the need for any specialised equipment. The speed and simplicity of our method makes it ideally suited for nucleic acid amplification-based applications both within and outside the laboratory, including limited resource settings such as remote field sites, developing countries, and teaching institutions.

Significance: Strigolactone hormones regulate many plant growth and developmental processes and are particularly important in regulating growth in response to nonoptimal conditions. Plants produce a range of bioactive strigolactone-like compounds, suggesting that the biosynthesis pathway is complex. Despite this complexity, only one type of enzyme, the MORE AXILLARY GROWTH1 (MAX1) cytochrome P450, has been attributed to the diversity of strigolactones. Using transcriptomics and reverse genetics, we discovered a previously uncharacterized gene that encodes a 2-oxoglutarate and Fe(II)-dependent dioxygenase involved in strigolactone production downstream of MAX1. Studies with the corresponding mutant have shown that previously identified strigolactone-type compounds inArabidopsisare not the major strigolactone-type shoot branching hormone in this model species.

Strigolactones act independently of auxin transport/canalization to inhibit bud outgrowth.