Plant viruses are important pathogens of agricultural and horticultural crops, causing economic losses of both yield and quality, and cannot be controlled by pesticides. The best means of control of plant virus diseases is to prevent the introduction of virus to the crop. However, in order to achieve prevention it is important to be able to detect and identify the virus, and the microarray offers a new means to do this.
Most existing methods of virus detection are either virus-specific, or detect a number of related viruses within one group, and are unable to detect viruses of other taxonomic groups. In contrast, the microarray will consist of thousands of oligonucleotides (short DNA ‘probes’), each derived from nucleic acid sequences conserved between the genomes (RNA or DNA) of members of a particular taxonomic group, or between isolates of a particular viral species. These ‘probes’ are spotted in an array to a support surface. Total nucleic acids from plants are enzymatically amplified (PCR) and labeled with fluorescent dyes and then overlaid on the microarray. Complementary viral sequences, if present, will hybridize (anneal) to their corresponding spotted viral ‘probe’. The array is then “read” to determine which, if any, viral complementary sequences were detected in the sample. The microarray will therefore be capable of detecting members of any taxonomic group represented by probes conserved within that group, with the goal of representing all known groups of plant viruses and virus-like pathogens.
The type of microarray to be developed has previously been utilized for the detection and identification of viruses affecting human populations, and was used to identify the virus responsible for SARS (Severe Acute Respiratory Syndrome; which first emerged in Asia in 2002) as a new member of a previously recognized group of viruses. Samples from SARS-affected patients were detected by probes specific for three distinct members of the Coronavirus group, indicating that the SARS virus belonged to the Coronavirus group, but that it was a previously uncharacterized member.
The same principles will allow detection of either characterized or previously unrecognized members of any plant virus group. The microarray will most likely be utilized in critical applications such as screening germplasm being introduced from other countries through quarantine facilities, and in the establishment of virus-tested nuclear stocks prior to large-scale vegetative propagation (e.g. for the introduction of a new cultivar).
'Universal' Plant Virus Microarray System: Diagram of Method
Complementary viral sequences, if present in sample, will hybridize (anneal) to their corresponding spotted viral ‘probe’
'Red' fluorescence indicates viral sequence present in sample
Last Updated May 1, 2009 2:34 PM
URL = http://www.usna.usda.gov/Research/MicroarrayGrant.html