Progress 09/28/07 to 09/27/11
Outputs
Progress Report Objectives (from AD-416) Develop sensitive, reliable methods for the detection and identification of plant viruses and viroids utilizing microarray techniques. Develop appropriate extraction and amplification methods suitable for application to diverse plant types. Develop appropriate data analysis techniques to allow development of a robust system for virus identification and discrimination between single and mixed infections. Approach (from AD-416) Develop multiple oligonucleotides representing conserved sequences for each recognized taxonomic grouping of plant viruses and viroids. Develop microarrays composed of such ologonucleotides for the detection and identification of diverse viruses and viroids, and data analysis methods for reproducible analysis of the results. Develop reagents, sampling procedures and protocols for the extraction amplification and labeling of nucleic acids from diverse plant types. The work falls under the auspices of, and is partially funded by, a reimbursable agreement between ARS and USAID project number 0210-22310- 004-11R. The ADODR has communicated with the Donald Danforth Plant Sciences Center (DDPSC) PI through email as well as receiving report updates to monitor progress. After the successful design and in-silico evaluation of the 9600 60-mer oligos, a Universal Plant Virus Microarray (UPVM) was printed by contact method on poly-llysine coated glass slides using the Linear-Servo (DeRisi model) arrayer. DDPSC printed two sets of UPVM arrays (261 slides each) and a set of UPVM sub-array slides (261 slides) for Geminiviruses, DNA satellites and viroids. Each slide consists of 16 blocks with 25 columns and 24 rows. After the post- processing of the newly printed slides, test hybridization was carried out to know the spot features using Axon 4000B laser scanner. Then the slides were distributed to three collaborating laboratories at USDA-ARS, Beltsville, MD; Oklahoma State University, Stillwater, OK and Cornell University at Ithaca, NY. DDPSC, like DemoPlantVirus array, optimized a single total RNA based sample preparation and labeling method using Cy dyes for both the DNA and RNA (-ve and +ve strand) plant viruses without amplification so that a sample infected with an unknown plant virus can be detected and identified by UPVM. DDPSC validated UPVM slides with six healthy plant samples such as tobacco, potato, tomato and more than 25 laboratory infected plant virus samples. Eighteen different virus species were successfully detected and identified. Most of the viruses were detected with relatively small amounts of RNA (2-3ug). For low-titer virus samples, increasing the total RNA (about 10x) resulted in detection while amplification with random PCR did not result in detection, possibly due to the lower signal-to-noise ratio in the template RNA. Analysis of the hybridization results at different probe levels such as family, genus and species level probes has been conducted. A data matrix of hybridization profiles of healthy and virus-infected samples for all of the 9600 probes has been prepared for masking/filtering the control and non-specific probes and for further statistical analysis. Analysis of the hybridization profile revealed that few probes are non-specific and are binding to most of the healthy and virus-infected samples e.g., Grapevine leaf-roll associated virus, Cherry small circular viroid, Persimon viroid and other viroid and DNA satellites probes. We also analyzed these results using computation algorithms like T-Predict (Taxonomy based E- Predict) and DetectiV. Detection based on visual intensities was well correlated with the E-Predict results. Iterative E-Predict algorithm was used for detection in the samples infected with more than one virus and for artificially mixed virus samples. Recently we obtained an old arrayer and are now planning to print more of UPVM slides and further validation of UPVM.
Impacts
(N/A)
Publications
|
Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Develop sensitive, reliable methods for the detection and identification of plant viruses and viroids utilizing microarray techniques. Develop appropriate extraction and amplification methods suitable for application to diverse plant types. Develop appropriate data analysis techniques to allow development of a robust system for virus identification and discrimination between single and mixed infections. Approach (from AD-416) Develop multiple oligonucleotides representing conserved sequences for each recognized taxonomic grouping of plant viruses and viroids. Develop microarrays composed of such ologonucleotides for the detection and identification of diverse viruses and viroids, and data analysis methods for reproducible analysis of the results. Develop reagents, sampling procedures and protocols for the extraction amplification and labeling of nucleic acids from diverse plant types. The ADODR has been in regular phone and email contact with the DDPSC as well as the ARS collaborator to receive updates and monitor progress. The first phase of this project saw the successful completion of using an existing microarray containing oligos for a few plant virusesiin the first year. For the second phase, the PI successfully made a DemoPlantVirus chip capable of recognizing 127 plant viruses. The third phase was to make a new PlantViro chip; to do the PI spent most of the second year designing and testing the selected oligos in silico. Before synthesizing these oligos the PI assessed them in silico for their capacity to identify known and unknown viruses of 12 representative families. A matrix of calculated binding energy between the 60 mer oligos and all of the target viruses was generated. Binding energy profiles of each target virus were compared with other viruses using the Pearson correlation coefficient. Statistical analysis determined the value of a species demarcation threshold for each of the 12 plant virus families. This greatly helps in identifying the new and/or unknown viruses. Using this approach, more than 94% of new isolates of begomoviruses were correctly identified. For all the 12 families studied here, there were no family level oligos that covered more than 75% of the species which could result in non-identification of the new species of a family. To address this concern the PI analyzed examples, such as the original unpruned oligo set (3.5 times more) for one family (Geminiviridae). Based on this analysis he added 164 generic oligos for different families. This approach was further extended to all the families of plant viruses to optimize the chances to identify unknown plant viruses using the UPVM. He is also developing an annotated complete reference genome database of all plant viruses to identify the known and unknown virus(es). Heisre building upon the already existing NCBI reference database of complete virus genomes and GenBank full length genomes. For the fabrication of full version of UPVM for more than 1350 plant viruses, satellites and viroids a total of ~33000 sense and reverse- compliment 60 mer candidate oligos consisting of both generic and specific oligos were designed using the algorithm. Due to the budget constraints for the purchase of these oligos only about ~10000 oligos were chosen for the synthesis. About 1000 glass slides coated with poly- lysine were prepared for fabrication of about 500 UPVM slides in the 2nd week of October 2010. The PI has also worked on a material transfer agreement with ATCC and other research institutes for a large scale testing (>500) and validation of the full version of the Universal Plant Virus Microarray (UPVM). To begin with we are checking the feasibility of this testing with a few representative samples, with the DemoPlantVirus chip first. He will extend that to the UPVM chip when available. If satisfactory, he will then process with the 500 ATCC samples and we will consider other collections outside our team.
Impacts
(N/A)
Publications
|
Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Develop sensitive, reliable methods for the detection and identification of plant viruses and viroids utilizing microarray techniques. Develop appropriate extraction and amplification methods suitable for application to diverse plant types. Develop appropriate data analysis techniques to allow development of a robust system for virus identification and discrimination between single and mixed infections. Approach (from AD-416) Develop multiple oligonucleotides representing conserved sequences for each recognized taxonomic grouping of plant viruses and viroids. Develop microarrays composed of such ologonucleotides for the detection and identification of diverse viruses and viroids, and data analysis methods for reproducible analysis of the results. Develop reagents, sampling procedures and protocols for the extraction amplification and labeling of nucleic acids from diverse plant types. Significant Activities that Support Special Target Populations This report serves to document research and activities carried out under a specific cooperative Agreement with the Donald Danforth Plant Science Center. The work falls under the auspices of and is partially funded by a reimbursable agreement between ARS and USAID (0210-22310-002-84R). Work continued with the plant ViroChip on the detection of a number of plant viruses using a range of different plant hosts to evaluate the potential difficulty to detect such viruses in plant extracts of a different nature. Our chip dedicated to plant viruses is using the same previously 70 mer oligos design that was printed in January 2009 to do this second phase of the work. The report below provides the detailed actions for that particular chip. The chip is efficiently working as all viruses tested have been detected with 100% accuracy for a range of viruses present at the labs of cooperators at ILTAB, USDA/ARS, University of Oklahoma and Cornell University. We have also been able to detect mixtures of viruses, either created on purpose, or discovered as contaminations. This shows a good use of this technology and it demonstrates that the ViroChip is working efficiently. So far we have a very small number of samples tested and the limitation comes from the fact that we still using the old 70 mer oligos of the original ViroChip. During the same period of time, collaborators at the University of Utah, Washington University and the DDPSC have been working at designing new oligos for all plant viruses (at 50 mer). The project has chosen 50mers instead of 70 mer to better accommodate smaller plant genomes and still have a fairly large number of oilgos per virus (6-12). The need to redesign the chip is also justified by the large increase in availability of virus sequences since the first chip was designed. Also, we are aiming to design generic oligos at the genus and family levels to detect unknown viruses in known families. The ADODR has been in regular phone and email contact with the DDPSC as well as the ARS collaborator to receive updates and monitor progress.
Impacts
(N/A)
Publications
|
|