In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using a cocktail of thermophilic proteinases and cell wall-degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This closed system delivers purified DNA with no cross-contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into nucleotide base called data locally in real-time, removing the need for high-specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and setup. To evaluate and validate capability of the system for unbiased pathogen identification by real-time sequencing in a farmer’s field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or destroy cassava crops. Eight hundred (800) million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint to its production. Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus-resistant or replacement planting. This case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, Internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making effective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis, including sample collection, processing and provisional sequencing results was complete in under 3 h. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation.

Virus diseases are among the main biotic factors constraining common bean (Phaseolus vulgaris L.) production in Tanzania. Disease management requires information on types, distribution, incidence, and genetic variation of the causal viruses, which is currently limited. Thus, a countrywide comprehensive survey was conducted. Use of a next-generation sequencing technique enabled simultaneous detection of 15 viruses belonging to 11 genera. De novo assembly resulted in many contigs, including complete or nearly complete sequences of Bean common mosaic virus (BCMV), Bean common mosaic necrosis virus (BCMNV), and Southern bean mosaic virus (SBMV). Some viruses (for example, SBMV and Tomato leaf curl Uganda virus-related begomovirus) were detected for the first time in common bean in Tanzania. Visually assessed virus-like disease incidence ranged from 0 to 98% but reverse-transcription polymerase chain reaction-based incidence of BCMV and BCMNV (7,756 samples) was mostly less than 40%. The Sanger-based nucleotide sequences encoding coat proteins of BCMV and BCMNV isolates were 90.2 to 100% and 97.1 to 100% identical to each other, respectively. Phylogenetic analysis showed that BCMV isolates were more diverse than BCMNV isolates. The information generated in this study will contribute to the development of molecular diagnostic tools and strategies for management of virus diseases nationally and internationally. [ ] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Background: Cassava leaf samples degrade quickly during storage and transportation from distant areas. Proper sampling and efficient, low-cost storage methods are critical for obtaining sufficient quality DNA and RNA for plant virus epidemiology and improving disease control understanding. This is useful when samples are collected from remote areas far from a laboratory or in developing countries where money and materials for virus diagnostics are scarce. Results: The effect of sample storage duration on nucleic acid (N.A.) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single N.A. extraction was optimized and tested alongside two existing CTAB-based methods (M1 and M2) for N.A. extraction from fresh and herbarium cassava leaves stored for; 1, 8, 26, and 56 months. The amount and quality of DNA and RNA were determined using Nanodrop 2000 c U.V.–vis Spectrophotometer and agarose gel electrophoreses. The sample degradation rate was estimated using a simple mathematical model in Matlab computational software. The results show no significant difference in mean DNA concentration between M1 and M2 but a significant difference between M3 and the other two methods at p < 0.005. The mean DNA concentration extracted using M3 was higher for 1 and 8 months of leave storage. M3 and M2 produced high concentrations at 26 and 56 months of leave storage. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at 8 and 26 months of leave storage and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean DNA quality between M1 and M2, but there was no significant difference between M3 and the other two methods at p < 0.005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at 0.0493 and 0.0521/month, while RNA was 0.0678 and 0.0744/month. Compared to the existing two methods, modified CTAB extracted enough high-quality N.A. in one-third the time of the existing two methods. Conclusion: Our method provides cost-effective, quick, and simple processing of fresh and dry samples, which will quicken and guide the decision on when and what type of sample to process for plant disease management and surveillance actions.

The United Nations has listed Zero Hunger as one of the 17 global sustainable development goals to end extreme poverty by 2030. Plant viruses are a major constraint to crop production globally causing an estimated $30 billion in damage ¹ leaving millions of people food insecure ² . In Africa, agriculture employs up to 50% of the workforce, yet only contributes 15% to the GDP on average ³ , suggesting that there is low productivity and limited value addition. This can be addressed through continued innovation in the fields of science and technology as suggested in the Science Agenda for Agriculture in Africa (S3A) ⁴ . Sustainable management of plant viruses and their associated vectors must include efficient diagnostics for surveillance, detection and identification to inform disease management, including the development and strategic deployment of virus resistant varieties. To date, researchers have been utilizing conventional methods such as; PCR, qPCR, high throughput sequencing (RNA-Seq, DNA-Seq) and Sanger sequencing for pathogen identification. However, these methods are both costly and time consuming, delaying timely control actions. The emergence of new tools for real-time diagnostics, such as the Oxford Nanopore MinION, have recently proven useful for early detection of Ebola ⁶ and Zika ^7,8 , even in low resourced laboratories. For the first time globally, the MinION portable pocket DNA sequencer was used to sequence whole plant virus genomes. We used this technology to identify the begomoviruses causing the devastating CMD which is ravaging smallholder farmers’ crops in sub-Saharan Africa. Cassava, a carbohydrate crop from which tapioca originates, is a major source of calories for over eight hundred (800) million people worldwide. With this technology, farmers struggling with diseased crops can take immediate, restorative action to improve their livelihoods based on information about the health of their plants, generated using a portable, real-time DNA sequencing device.

Background: Cassava leaf samples can degrade quickly during storage and transportation from distant areas. Proper sampling and the use of efficient cheap storage methods are critical to obtain sufficient quality of DNA and RNA for plant virus epidemiology and to improve understanding on disease control. This is practical when samples are collected from isolated zones distant from a laboratory, or in developing countries that lack a supply of money and materials for virus diagnostics. Results: The effect of sample storage duration on nucleic acid (NA) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single NA extraction was optimized and evaluated together with two existing CTAB-based methods (M1 and M2) for extraction of NA from fresh and herbarium cassava leaves stored at; 1, 8, 26, and 56 months. The quantity of DNA and quality of both DNA and RNA were evaluated using Nanodrop 2000c UV–vis Spectrophotometer and agarose gel electrophoreses, and the rate of sample degradation was estimated using a simple mathematical model in Matlab computational software. The results show that there is no significant difference between M1 and M2 in the mean concentration of DNA but there was a significant difference between M3 and the other two methods at p < 0.005. The mean concentration of DNA extracted using M3 was higher at 1 and 8 months of age. M3 and M2 produced high concentrations at the age of 26 and 56 months. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at the age of 8 and 26 months and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean quality of DNA between M1 and M2 but there was no significant difference between M3 and the other two methods at p < 0.005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at the rate of 0.0493 and 0.0521/month while RNA was 0.0678 and 0.0744/month. Modified CTAB extracted a sufficient amount of NA of high quality for a third of the time (28/95min) compared to the existing two methods. Conclusion: Our method will provide the cost-effective, quick, and simple processing of fresh and dry samples which will quicken and guide the decision process on when and what type of sample to process for plant disease management and surveillance actions.

Unpredictable emerging and re-emerging of Mpox virus (MPXV) outbreaks account for urgent interventions. While no drug has been approved for Mpox treatment, repurposing of nucleoside analog drugs such as tecovirimat and cidofovir is the current focus of many intervention initiatives. Intrigued by their molecular structural similarities, we hypothesized that acyclovir, allopurinol, and B vitamins, which structurally resemble tecovirimat and cidofovir, could be competitive inhibitors of the MPXV DNA polymerase, abrogating DNA synthesis viral multiplication. We applied computational molecular docking experiments to investigate this hypothesis. Our results revealed that acyclovir fits exactly into the binding cavity for cidofovir and interacts with the same MPXV DNA polymerase amino acid residues with comparatively similar binding energy. We also found that oxipurinol (a metabolite of allopurinol) expresses higher affinity for MPXV DNA polymerase than cidofovir. Interestingly, the B vitamins riboflavin and folic acid outcompeted cidofovir, acyclovir and deoxyribonucleoside triphosphates (dNTPs), the natural substrates for DNA polymerase. From these ligand-receptor interactions we infer that acyclovir, oxipurinol and B vitamins are competitive inhibitors, targeting the active rather than the allosteric site of MPXV DNA polymerase. Our findings provide a novel insight into the B vitamin’s mode of action, emphasizing their antiviral potential via direct hijacking of the viral DNA replication machinery. While B vitamins are easy to find and cheaper, wet laboratory studies equipped with bioinformatics capacity could validate our findings to drive clinical trials. Once well established, B vitamins can be a cheap and reliable venture for pandemic preparedness for viral outbreaks.