Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): To develop a universal plant virus micro array for detection and identification of plant viruses. Approach (from AD-416): Develop microarrray containing probes representing all known plant viruses. Most virus detection methods are specific for a single virus or closely- related viruses, and are unable to detect viruses that have not previously been characterized. It is thus possible to detect only the specific viruses tested for, and not possible to identify all components of the mixed infections common in vegetatively-propagated plants. It is highly desirable to have a method that can detect any virus, including previously uncharacterized viruses, and to identify all components of mixed infections. ARS researchers at Beltsville, MD and collaborators at the Danforth Science Center, Washington University, University of Utah, Oklahoma State University, and Cornell University, have developed a Universal Plant Virus Microarray (UPVM) with funding from the USDA NRI Plant Biosecurity program (grant no. 2009-55605-05023). At Beltsville work continued to optimize the ABC-DSN protocol and expand the range of viruses and hosts tested on the UPVM. The ABC-DSN protocol enriches for viral RNA by removing most ribosomal RNAs through subtractive hybridization. This process has allowed us to detect several low-titer viruses that could not be identified by direct labeling of cDNA or standard ABC amplification. The improved ABC-DSN protocol is more sensitive and easier to perform. Since the previous update we have identified viruses from over 9 families (12 genera) from more than 15 host families (19 host genera). Included were several single blind tests (i.e. virus identity unknown to the tester) in which the expected viruses were detected by the UPVM. In other tests mixed infections were identified in hosts thought to only have a single infection, and results confirmed by PCR detection of the additional viruses. We are continuing to process batches of samples from APHIS, including infections in sweet potato, cherry, and blackberry. At the Danforth Science Center additional virus samples from grapevines, hops, fruit trees, potato, sweet potato, cassava , cereals and other sources were prepared and assayed, with approximately 280 hybridizations, including some previously untested genera. These results, including iterative T-predict analysis at the University of Utah, aid the interpretation of UPVM hybridization results. Additional printings of the UPVM were made using the modified print layout. At Cornell University some UPVM probes were utilized on macroarrays for detection of viruses affecting grapes, and to detect two viruses previously unreported in the northeastern U.S. At Oklahoma State University UPVM probes were used to screen high-throughput sequencing data, aiding the identification of viral sequences from one of three samples, and positive plant controls but no viral sequences in two other samples. Several cucurbit samples were tested by hybridization to the UPVM; however the viruses detected in these samples differed from those detected by serological assays. This information will be of most immediate application to the UPVM collaborators, but will also be of value to regulatory agencies, plant diagnostic clinics, germplasm repositories, and producers operating plant certification schemes.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): To develop a universal plant virus micro array for detection and identification of plant viruses. Approach (from AD-416): Develop microarrray containing probes representing all known plant viruses. Most virus detection methods are specific for a single virus or closely- related viruses, and are unable to detect viruses that have not previously been characterized. It is thus possible to detect only the specific viruses tested for, and not possible to identify all components of the mixed infections common in vegetatively-propagated plants. It is highly desirable to have a method that can detect any virus, including previously uncharacterized viruses, and to identify all components of mixed infections. ARS researchers at Beltsville, MD in collaboration with scientists at the Danforth Science Center, Washington University, University of Utah, Oklahoma State University, and Cornell University, have developed a Universal Plant Virus Microarray (UPVM) with funding from the USDA NRI Plant Biosecurity program (grant no. 2009-55605-05023). Work at Beltsville has concentrated on increasing the sensitivity of detection of viruses occurring at low titer, as initial protocols (direct labeling of cDNA; or unbiased ABC amplification) did not yield reliable results. A subtractive hybridization protocol utilizing a duplex specific nuclease to reduce high-copy sequences such as ribosomal RNA, from samples, followed by the ABC amplification protocol, has allowed detection of several viruses not detected by previous methods. This protocol is significantly cheaper than subtractive hybridization using commercially-available kits. The duplex specific nuclease method has also allowed detection of a mixed infection in a sample previously known to be infected by only one virus. Collaborators at the Danforth Science Center have also increased sensitivity either by using significantly higher levels of sample nucleic acids, or by use of commercial subtractive hybridization kits; they also evaluated both diseased and healthy samples of several plant species in order to identify non-specific reactions of several of the 60-mer UPVM probes. These results, and improvements to the iterative T-predict analysis at the University of Utah, aid the interpretation of UPVM hybridization results. Additional printings of the UPVM were made using a different print layout, reducing the print time by about half. Extracts of virus-infected grapevines were prepared at Cornell University and sent to the Danforth Science Center for testing. At Oklahoma State University the UPVM probes were used to screen high- throughput sequencing data, aiding the identification of viral sequences with 100% specificity, over 80% accuracy of identification, and no false positives. Some of these results were presented at the UPVM Workshop and BARD Workshop �Microarrays and Next-Generation Sequencing for Detection and Identification of Plant Viruses� held at Beltsville in November 2011. This information will be of most immediate application to the UPVM collaborators, but will also be of value to regulatory agencies, plant diagnostic clinics, germplasm repositories, and producers operating plant certification schemes.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) To develop a universal plant virus micro array for detection and identification of plant viruses. Approach (from AD-416) Develop microarrray containing probes representing all known plant viruses. Most virus detection methods are specific for a single virus or closely- related viruses, and are unable to detect viruses that have not previously been characterized. It is thus possible to detect only the specific viruses tested for, and not possible to identify all components of the mixed infections common in vegetatively-propagated plants. It is highly desirable to have a method that can detect any virus, including previously uncharacterized viruses, and to identify all components of mixed infections. ARS researchers at Beltsville, MD have, with funding from the USDA NRI Plant Biosecurity program (#2009-55605-05023), and in collaboration with scientists at the Danforth Science Center, Washington University, University of Utah, Oklahoma State University, and Cornell University, developed a Universal Plant Virus Microarray (UPVM). The UPVM has 9,600 oligonucleotides representing sequences conserved among members of an individual virus species, among members of a viral genus, or among members of a viral family (from genomic sequences available in public databases as of December 2009); and 44 highly conserved plant genes as controls. Oligonucleotide design and selection was based on E-predict, by a University of Utah collaborator. Other software (UChip and T-Predict), adapted specifically for the UPVM, is used to analyze results from hybridization of nucleic acids extracted from infected plant samples. We have demonstrated that the UPVM can detect a number of high-titered characterized plant viruses, including differentiation of the components of mixed infections, and work to validate the UPVM for a larger number of viruses continues. Work is in progress to develop suitable non-specific amplification techniques to allow detection of viruses occurring at low concentration in host tissues. Some previously undescribed viruses were correctly assigned to the taxonomic group indicated by independent genome sequencing, demonstrating the potential of the UPVM to detect previously unknown plant viruses. Beltsville ARS researchers developed a method (the �CKC� method) suitable for the rapid and routine extraction of total nucleic acids from a wide variety of plant tissues for use with the UPVM. The method has been tested on over 60 species from 35 diverse plant families, including species rich in polysaccharides, phenolics, and latex. The UPVM is expected to benefit regulatory agencies, germplasm repositories, and breeders or others introducing new plant materials. Communications to monitor progress were carried out by e-mail, conference calls, a site visit between the various partners, by a meeting during the Annual meeting of the American Phytopathological Society, a meeting at the NRI Plant Biosecurity program directors meeting, and by written and oral reports to the NRI Plant Biosecurity Program.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) To develop a universal plant virus micro array for detection and identification of plant viruses. Approach (from AD-416) Develop microarrray containing probes representing all known plant viruses. This research was carried out under a National Research Initiative Plant Biosecurity Program grant, in collaboration with scientists at the Donald Danforth Plant Science Center, Washington University, the University of Utah, Oklahoma State University, and Cornell University. The goals are to able to detect any plant virus in extracts of infected plants, and to identify previously characterized viruses to the species level, or previously uncharacterized virus to at least the viral family or genus level. Progress was made towards development of a robust and reliable method for total nucleic acid extraction applicable to diverse plant species. The best strategy to control viral pathogens in plants is rapid identification and detection in quarantine, breeding programs, certification, and production. The various genera of plant viruses have different types of nucleic acid as their genome; either single-stranded or double-stranded DNA, or single-stranded or double stranded RNA. ARS researchers at Beltsville, MD are working with collaborators to develop a Universal Plant Virus Microarray for the detection and identification of plant viruses and viroids. Molecular techniques such as polymerase chain reaction (PCR)-based tests, and microarrays, can be fast and sensitive, and are increasingly used to detect genetic materials of these pathogens. A significant challenge in using these techniques, however, is the need to prepare plant samples or extracts containing these nucleic acids, but free of other compounds that occur in plant cells, such as oligosaccharides and polyphenols, that can interfere with the pathogen detection technique. To enable rapid detection of plant viruses and viroids from any potential plant host, a method for preparing total nucleic acids is required, which will yield both RNA and DNA of adequate yield and purity for detection techniques. ARS scientists at Beltsville, MD have developed a robust and reliable method of nucleic acid extraction for the microarray-based detection of diverse plant pathogens, including the viruses and viroids that are the target of the Universal Plant Virus Microarray. The method developed has been optimized for speed and purity of the nucleic acids in order to facilitate sample throughput. The method has been tested successfully against a diverse range of plant material known to contain significant levels of compounds that potentially inhibit nucleic acid amplification and labeling. The resulting total nucleic acid extracts were evaluated for purity, and tested by amplification of viral sequences known to be present. Samples prepared by this method will be utilized for validation of the Universal Plant Virus Microarray. This information will also be useful to those who work in quarantine and certification programs that need to test valuable plant material for pathogens. Communications to monitor progress were carried out by e-mail and conference calls between the various partners, by a group meeting during the Annual meeting of the American Phytopathological Society, and by written and oral reports to the NRI Plant Biosecurity Program.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) To develop a universal plant virus micro array for detection and identification of plant viruses. Approach (from AD-416) Develop microarrray containing probes representing all known plant viruses. Significant Activities that Support Special Target Populations A project to develop a Universal Plant Virus Microarray has been initiated as a collaborative effort between the Floral and Nursery Plants Research Unit, ARS; the Donald Danforth Plant Science Center; Washington University; the University of Utah; Oklahoma State University, and Cornell University. The goals are to be able to detect any plant virus in extracts of infected plants, and to identify previously characterized viruses to the species level, or previously uncharacterized virus to at least the viral family or genus level. Prior to development of the full microarray, the system is being validated for approximately 50 characterized viruses represented by a total of 750 virus-specific oligonucleotides, plus control host genes, on a �MiniPlantViroChip� array. To date all but one of the virus isolates tested have been successfully detected with the majority of oligonucleotides representing the virus in question, without amplification of the samples. The tested samples represent viruses with both RNA and DNA genomes, including members of the families Geminiviridae and Caulimoviridae (DNA), as well as species in the Bromoviridae, Flexiviridae, Potyviridae, and genus Tobamovirus (RNA), including multiple strains of some viruses. These results demonstrate the specificity and sensitivity of the array over a wide dynamic range of probe intensities, and form a strong background for development of the full scale Universal array. The Universal Plant Virus Microarray will be of value to diagnosticians in the National Plant Diagnostic Network, plant introduction and quarantine facilities, clean stock virus certification programs, and plant propagators introducing new varieties. Research activities conducted under this agreement were monitored by regular email and phone communication and in person at scientific meetings.
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