Final Report for GNE11-019
Infectious plant pathogens represent an expensive source of crop loss, but modern methods to monitor and identify infectious agents typically require elaborate laboratory infrastructure, trained personnel, and reagents that may need special handling and storage. In contrast, the ideal plant pathogen detection method would be would be straightforward to use and capable of detection on-site. We have developed an approach for nucleic acids detection that involves only a simple enzymatic reaction and does not require expensive equipment. As a proof of concept, we have successfully detected pathogen from tobacco leaves infected with the cucumber mosaic virus using an inexpensive assay that can be performed simply at a constant temperature. This method represents a dramatic improvement for on-site nucleic acid detection, which could eventually be carried out directly by farmers and enable earlier detection of diseased crops.
Plant pathogens are a costly burden for United States agriculture, causing an estimated $33 billion worth of damage annually. This expense represents a significant challenge for the sustainability of US agriculture and food systems. Central to the control of plant pathogens is the reliable and on-site identification of the causal agents. In order to more effectively protect our agriculture and food systems from infectious disease, diagnostic tests should be fast, inexpensive, sensitive, and performed on-site without specially trained personnel. Farmers would greatly benefit from direct and hands-on access to accurate diagnostic technologies for identifying plant pathogens. The ability to diagnose plant pathogens at an early stage of infection would enable earlier and more cost effective treatment options, and would also allow better monitoring for recording the spread of crop infections.
While there have been intense research on point-of-care (POC) biosensors towards medical diagnostics, detection systems that are specifically designed for agriculture and food systems in general and plant pathogen in particular has been lacking. A wide variety of effective technologies exist for pathogen detection, however it remains challenging to implement modern detection methods in a practical way that can be performed on-site. Typical methods for plant pathogen detection, such as would be used in a modern plant pathology laboratory, require special training and are not practical for on-site use.[2, 3] In particular, most nucleic acid detection methods are based on polymerase chain reaction (PCR) which requires special equipment capable of precisely controlling the temperature.
In contrast to established approaches, we have developed a nucleic acid detection method that is based on isothermal (constant temperature) amplification of the target nucleic acids within a test sample. By implementing our assay on-site, we aimed to achieve the following:
Reduced time-to-answer: Many lab-based diagnostic tests for plant pathogens require samples to be shipped to a central location. In many situations this approach is inconvenient and inefficient, and causes delays in treatment while the sample is transported to a central facility. This delay prevents timely action that could prevent spread of the infection.
Increased flexibility: The diagnosis of infectious diseases is currently a complex task with no “one size fits all” solution. For instance, immunologically-based assays require the development of antibodies for every unique antigen. In contrast, nucleic acid-based test is generally applicable for all pathogens. Nucleic acid tests rely on DNA targets, for which sequences are known for almost all relevant pathogens. In this manner, by reducing variation in the assays required for a diagnosis, the detection process can be simplified.
Reduced cost: Current methods for nucleic acid detection require additional investment in specialized equipment. In contrast, our assay avoids the use of complicated instruments or machines, which enables a much lower material cost per assay.
To achieve these advantages, our project focused on the application of nucleic acid detection methods to develop a platform that is compatible with on-site detection of crop viruses. Our laboratory has previously developed novel DNA-based “fluorescence nanobarcodes” that could rapidly identify multiple pathogens simultaneously in a single assay. Each DNA-based fluorescence nanobarcodes contains a built-in, color-ratio code for the simultaneous sensing of multiple pathogenic DNA.[4-6] Here, we worked to extend our nanotechnology-based approaches to achieve on-site crop pathogen detection.
This proposal focused on the detection of crop viruses. However, over the long term, the proposed research could be easily extended to the detection of virtually any infectious disease since every pathogen has a genome, which can serve as a universal pathogen identifier. The proposed technology would have tremendous impact on a range of applications in agriculture and food systems, including on-site detection of infectious diseases in animals, as well as monitoring of food systems.
- Pimentel, D., Zuniga, R. & Morrison, D. Update on the environmental and economic costs associated with alien-invasive species in the United States. Ecological Economics 52, 273-288 (2005).
- Ngom, B., Guo, Y.C., Wang, X.L. & Bi, D.R. Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review. Analytical and Bioanalytical Chemistry 397, 1113-1135 (2010).
- Posthuma-Trumpie, G.A., Korf, J. & van Amerongen, A. Lateral flow (immuno) assay: its strengths, weaknesses, opportunities and threats. A literature survey. Analytical and Bioanalytical Chemistry 393, 569-582 (2009).
- Li, Y., Cu, Y.T.H. & Luo, D. Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes. Nature Biotechnology 23, 885-889 (2005).
- Um, S.H., Lee, J.B., Kwon, S.Y., Li, Y. & Luo, D. Dendrimer-like DNA-based fluorescence nanobarcodes. Nature Protocols 1, 995-1000 (2006).
- Lee, J.B. et al. Multifunctional nanoarchitectures from DNA-based ABC monomers. Nature Nanotechnology 4, 430-436 (2009).
The overall aim of this project was to develop a DNA nanotechnology-based platform for the on-site detection of crop viruses. The proposed detection system was to have the following characteristics: rapid, inexpensive, requiring no additional equipment, and easy to use for the non-specialist. Our plan of work consisted of two main objectives:
(1) Objective 1: Apply our DNA nanobarcode system to detect a panel of common plant pathogens with high sensitivity and specificity;
During the course of this project we focused on two representative plant pathogens: cucumber mosaic virus and tobacco yellow leaf curl virus. After our initial phases of testing, we encountered difficulties with our original approach utilizing DNA nanobarcodes. Although our strategy was effective with short synthetic DNA sequences, performance was poor with longer, more realistic RNA targets. As a result, we transitioned to an enzyme-based system using branching rolling chain amplification (RCA) that allowed us to achieved good performance with real samples while also improving our sensitivity. Using this method we achieved detection of pathogen RNA from plant leaves infected with the Cucumber Mosaic Virus. In future work, we expect to generalize our approach for testing with various other common plant pathogens as well as additional types of crops and samples.
(2) Objective 2: Integrate the sample preparation and signal readout modules into the platform and evaluate the robustness of our technology by testing with real plant tissue samples.
The development of our detection technology focused on the integration of three independent modules: (i) a sample preparation module; (ii) a target recognition and detection module; and (iii) a read-out module. Throughout our project we considered all three components; however, the main supporting objective for our proposal was focused on the target recognition and signal amplification modules, as we believed this was the most critical component and required the most development. In contrast, the other two components (sample preparation and signal readout), have been studied extensively. For example, commercial kits are widely available for the extraction of nucleic acids, and inexpensive equipment for fluorescence detection can be readily obtained for the read-out module. Using established methods for RNA isolation and gel electrophoresis as a readout method, we successfully detected target from real plant tissue samples.
Our research plan focused on development of an assay suitable for on-site detection, with a development strategy based on incremental improvement of our detection method to achieve a fully functional assay. To this end, we began our research project by detecting synthetic nucleic acid targets (commercially available DNA) that were readily available and easy to work with. Once we had demonstrated the detection with these synthetic targets, we gradually tested with sample types that were increasingly complex. Although we originally intended to develop a prototypical portable device for performing the assay, our assay development took longer than anticipated and therefore our efforts focused entirely on improving the assay performance.
An overview of our initial enzyme-free detection method is portrayed in Figure 1. Briefly, we prepared DNA nanostructures with recognition probes that can bind to target pathogen nucleic acid. This detection strategy was based on the crosslinking ability of branched DNA as described in related publications from our lab.[1,2] These DNA nanostructures could be linked together to form large aggregates only in the presence of a specific nucleic acid target sequence. The resulting aggregates could then be identified via fluorescence read-out. Importantly, this detection method is general for any pathogen, so it can be applied to other agriculturally-relevant fields such as detecting infectious diseases of animals and for food production.
During the course of our project, we realized an enzyme-based strategy was necessary to achieve adequate limit of detection. In our initial project proposal, we had concerns about using enzymes in our diagnostic test, including (1) cost, (2) stability for storage, and (3) sensitivity to environmental conditions (e.g. temperature) that may result in variable test performance. However, after testing various strategies for enzyme-free detection as well as testing real samples, we determined that an enzyme-based strategy would be necessary. Furthermore, recent developments in enzyme-stabilizing technologies have made portable testing with enzymes increasingly feasible.
To facilitate development of the diagnostic system, we drew on an ongoing collaboration with Prof. Keith Perry from the Department of Plant Pathology at Cornell University. Prof. Perry is an expert in crop pathology and guided us in selecting probes and providing samples. We focused on the detection of two representative plant viruses, one with a DNA genome and one with an RNA genome. The DNA virus Tomato yellow leaf curl virus (TYLCV) has a bipartite genome consisting of two single-stranded (ss) DNAs, each 2.6 kb in size. The RNA virus cucumber mosaic virus (CMV) has a tripartite genome of three positive sense ssRNAs. Tomato and tobacco plants were used as hosts. Both viruses have been worked on extensively as model systems, with fully sequenced genomes of many isolates. These viruses are also economically important pathogens worldwide. For detection of these viruses, we used specific oligonucleotide probes based on Prof. Perry’s previous work. These probes had already been described and validated.[6,7,8] For CMV, probes were available to recognize all three of the target genomic RNAs and thus provide flexibility for selecting sequences. Similarly, TYLCV probes were available that were both virus- and family- specific; these probes were designed to detect either the genomic DNA or RNA transcription products from the virus. In addition, a subset of these probes had already been shown to be effective in the detection of TYLCV in field samples.
Initial experiments were performed using extracted DNA or complementary DNA (cDNAs) prepared from RNA extracts. In array or PCR-based detection strategies, viral RNAs are typically detected by first converting them into cDNAs. In our assays, we often used unpurified RNAs because this represented a more direct approach for detection. However, initial experiments were performed using purified RNAs to evaluate methods and optimize assay conditions.[1,7]
- Lee, J.B. et al. “Multifunctional nanoarchitectures from DNA-based ABC monomers.” Nature Nanotechnology, 4, 430-436 (2009).
- Li, Y., Cu, Y.T.H. & Luo, D. “Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes.” Nature Biotechnology, 23, 885-889 (2005).
- Y. Sun, J. Høgberg, T. Christine, L. Florian, L. G. Monsalve, S. Rodriguez, C. Cao, A. Wolff, J. M. Ruano-Lopez, and D. D. Bang. “Pre-storage of gelified reagents in a lab-on-a-foil system for rapid nucleic acid analysis.” Lab Chip, 13, 1509–14 (2013).
- Gafni, Y. Tomato yellow leaf curl virus, the intracellular dynamics of a plant DNA virus. Molecular Plant Pathology 4, 9-15 (2003).
- Palukaitis, P. & Garcia-Arenal, F. Cucumber Mosaic Virus. Advances in Virus Research 62, 241-323 (2003).
- Agindotan, B. & Perry, K.L. Macroarray detection of eleven potato-infecting viruses and Potato spindle tuber viroid. Plant Disease 92, 730-740 (2008).
- Agindotan, B. & Perry, K.L. Macroarray detection of plant RNA viruses using randomly primed and amplified complementary DNAs from infected plants. Phytopathology 97, 119-127 (2007).
- Perry, K.L. & Lu, X. A tospovirus new to North America: Virus detection and discovery through the use of a macroarray for viruses of solanaceous crops. Phytopathology 100, S100 (2010).
Our primary accomplishment was the development of an isothermal nucleic acid detection method for plant pathogen detection. The main goal was to determine the sensitivity and specificity of our branched DNA detection approach using progressively more realistic mock plant pathogen samples, starting with relatively short synthetic DNA sequences from pathogen DNA, with the final goal of testing our approach with real diseased plant specimens.
We demonstrated all results using gel electrophoresis as a readout method because this was the most convenient method to use in the laboratory. For on-site implementation, electrophoresis readouts can be readily translated into a portable readout that is more suitable for on-site detection.
For initial testing, we selected a 100 base region of the cucumber mosaic virus (CMV) to act as a mock pathogen sample. We designed corresponding probes to form aggregates specifically in the presence of the DNA sequence. In these initial tests with short synthetic DNA sequences, we found that our assay was effective in binding most of the branched YDNA probe present in the solution (Figure 2). As a next step, we tested our assay with a 2,000 base RNA representing a realistic sample type. This sequence was RNA-3 from the CMV genome (GenBank accession number NC_001440.1) and was provided by our collaborator Prof. Keith Perry. Working with these samples, we obtained a negative result in which aggregation was not observed even when the target was present (Figure 3). This suggests that the YDNA probes did not bind successfully to the long RNA-3 target. To resolve this problem, we attempted to increase the favorability of the binding process by adjusting the protocol of our detection approach. We systematically varied several key parameters of our assay, including the molar ratio between target and probe (Figure 4), and the amount of magnesium (Figure 5). The amount of magnesium was expected to influence the extent of secondary structure of the RNA, which could potentially obstruct hybridization and interfere with our assay. Unfortunately, these parameters did not result in increased aggregation. We also tested a comparison between target RNA and DNA, and observed aggregation was much more successful using DNA compared to RNA (Figure 6). This suggests that our original approach was most effective for DNA targets, which represented a major hurdle for our assay since most plant viruses have RNA genomes.
To address this limitation and also increase the sensitivity of our assay, we decided to introduce an enzymatic amplification step in which only a portion of the pathogen nucleic acid was amplified. The addition of an enzymatic amplification step to our assay represented a change to our original plan, but one that was readily integrated into our existing protocols. The amplification of target nucleic acid allowed us to more easily sense the presence of pathogenic nucleic acid, and also overcame the original limitation of our assay based on target length. We continued to focus on the use of branched DNA as a novel detection strategy, and to focus on plant disease CMV as a test pathogen. The addition of an enzymatic amplification step made our strategy more feasible for real-world implementation.
Our enzyme-based method was inspired by recent publications, including one publication from our laboratory, which used isothermal rolling chain amplification (RCA). This resulted in a dramatic increase in assay sensitivity.[1-3] We designed a template sequence composed of single-stranded DNA which enabled amplification only in the presence of target (Figure 7). Based on the performance of similar strategies, we anticipated that our assay could achieve femtomolar level sensitivity. In addition to pathogen sensing potential, this approach will be useful for sensitive detection of microRNA, which has gained significant attention recently for being an excellent indicator of disease states.
For initial testing, we demonstrated our enzyme-based method with short purified RNA. This RNA was obtained from real Cucumber Mosaic Virus samples; however, it was processed into a shorter form that would be easier to detect than the full-length viral RNA as it exists in nature. As shown in Figure 8, our enzyme-based reaction gave the expected product ONLY in the presence of the pathogen target, indicating that RCA has the ability for target specific amplification.
After this proof of concept, we isolated RNA from real samples. As shown in Figure 9, we worked directly with CMV infected leaves. Using these leaves, we experimented with isolation procedures to extract the necessary nucleic acids for our detection assay. We selected isolation methods that could successfully extract RNA in high yield and with minimal degradation (Figure 10). Notably, this process resulted in a mixture of both plant and viral RNA. The viral RNA was then specifically amplified via our enzymatic process to obtain a diagnostic result (Figure 11).
One critical consideration for our process was the type of enzyme used for amplification. For initial testing we used the enzymes DNA ligase and Taq polymerase. For further testing, we tested alternative enzymes in order to determine the best choice based on multiple parameters including (1) cost, (2) stability, and (3) performance of the enzyme. We tested two different polymerases under standard conditions. As shown in Fig. 12, the reaction worked for both enzymes as indicated by the high bands in the gel electrophoreses, but the Deep Vent Polymerase was more stable (which may correspond to increased stability for on-site detection) and less expensive. In addition, we made efforts to simply the assay methodology by comparing a one- and two-step reaction. Interestingly, we observed that the assay performed just as well with a one-step simplified procedure (compatible with on-site detection) compared with a more complicated two-step process (inconvenient for on-site detection), as shown in Figure 13.
1. Neubacher and Arenz. “Rolling-Circle Amplification: Unshared Advantages in miRNA Detection.” ChemBioChem, 10, 1289 – 1291 (2009).
2. Cheng, et al. “Highly Sensitive Determination of microRNA Using Target-Primed and Branched Rolling-Circle Amplification.” Angew. Chem. Int. Ed., 48, 3268 – 3272 (2009).
3. Lee, et al. “A Mechanical Metamaterial Made From a DNA Hydrogel.” Nature Nanotechnology, 7, 816 – 820 (2012).
We envisioned that this on-site detection would primarily be used by farmers for monitoring crop diseases, but it could also have impact on extension groups as well as other plant growers (horticulturists, plant nurseries, distributors of plant products). Since every pathogen has genetic material, nucleic acid detection strategies are extremely general and could be applied to many different settings – for example, identifying disease in livestock or sensing infectious pathogens in food supplies.
Education & Outreach Activities and Participation Summary
Based on this work for branched DNA structures for detection, we have published on aspects of our research in scientific journals, including Nanoscale, Angewandte Chemie, and Journal of the American Chemical Society.[1,2,3] Additional papers are in preparation. These publications will promote continued work with on-site diagnostics and nucleic acid based detection within the scientific community. In addition, an oral presentation was given, titled “Branched PCR using Thermostable DNA Nanostructures as Primers”, at the Biological and Environmental Engineering Research Symposium at Cornell University in 2013. An undergraduate student was also recruited to the project; this student was trained and assisted with the project throughout the summer of 2012.
 M. R. Hartman, R. C. H. Ruiz, S. Hamada, C. Xu, K. G. Yancey, Y. Yu, W. Han, and D. Luo, “Point-of-care nucleic acid detection using nanotechnology.,” Nanoscale, 5, 21, 10141–10154 (2013).
 M. R. Hartman, D. Yang, T. N. N. Tran, K. Lee, J. S. Kahn, P. Kiatwuthinon, K. G. Yancey, O. Trotsenko, S. Minko, and D. Luo, “Thermostable Branched DNA Nanostructures as Modular Primers for Polymerase Chain Reaction.,” Angew Chem Int Ed Engl, 52, 33, 8699–702 (2013).
 T. N. N. Tran, J. Cui, M. R. Hartman, S. Peng, H. Funabashi, F. Duan, D. Yang, J. C. March, J. T. Lis, H. Cui, and D. Luo, “A Universal DNA-Based Protein Detection System.,” J Am Chem Soc, 135, 38, 14008–11 (2013).
We believe the eventually impact of more timely diagnosis for infectious plant pathogens will eventually have extremely large impact due to the devastating amount of crop loss arising from infectious disease. However, as our assay is not yet fully implemented or disseminated, we cannot claim any concrete or quantitative improvements in terms of economic benefits or sustainability.
Due to our focus on the earlier stages of assay development, we did not engage directly with farmers. We believe our project could promote the adoption of on-site detection, which will be important for farmers to immediately detect and identify diseases, and thus take necessary preventative steps to minimize the infection and avoid spread of the disease. With further development of our assay, we believe engagement with farmers would be necessary in order to seek their expert feedback about practical implementation of our assay.
Areas needing additional study
Future work in this area will involve further improvement of our nucleic acid assay, including: (1) generalizing our assay for broader use by testing and validation with additional disease types and species of crops; (2) further optimization of key parameters of our method (such as concentrations of enzymes and chemical reagents); (3) development of a prototypical self-contained device, allowing for increased portability and ease-of-use for our assay. Furthermore, in order to facilitate ease-of-use for on-site crop monitoring, we expect diagnostic methods such as the method proposed here can eventually be implemented into automatic sensor devices that could ultimately monitor crops in an entirely automated fashion.