Detection of odontoglossum ringspot virus in inoculated orchid leaf tissue using SYBR green real-time RT-PCR

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Haaning, Allison M.
Vann, Carolyn N.
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Thesis (M.S.)
Department of Biology
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Odontoglossum ringspot virus (ORSV) is one of the most prevalent orchid viruses that infects greenhouse-grown orchids worldwide. In order to prevent the spread of viruses in greenhouses and to cultivate clones from virus-free mother plants, it is necessary to develop a more sensitive technique for the detection of viruses in orchids. SYBR green real-time RT-PCR is a highly sensitive technique that can specifically detect ORSV in orchid tissue. By harvesting tissue at the inoculation site and at specific distances from the inoculation site at different times past inoculation, this technique can also be used to study the rate of spread of ORSV in orchids. Orchid clones were inoculated with ORSV and other clones were mock-inoculated with molecular grade water. Leaf tissue was harvested from the ORSV-inoculated and mock-inoculated clones at the site of inoculation and at specific distances from this site at 16 h, 24 h, and 72 h past inoculation. Total RNA was extracted from the harvested tissue. Competitive RTPCR was going to be used for the quantification and detection of ORSV in the samples, but attempts at cloning an ORSV fragment into a vector in order to form a competitive standard were unsuccessful. Instead, a highly sensitive qualitative approach called SYBR green real-time RT-PCR was used for the detection of ORSV. ORSV was detected in all virus-inoculated orchids, except for one. Therefore, all of the ORSV inoculated plants except for one were infected with the virus. Unexpectedly, ORSV was also detected in all of the mock-inoculated orchids. Most likely the orchids were previously infected with ORSV, but the viral titer was too low to be detected by commercial techniques. However, there is a small possibility that the orchids were contaminated during experimentation, despite careful technique. The rate of spread of the virus could not be studied because the mock-inoculated samples also contained the virus. Although viral amplification was demonstrated in the mock-inoculated plants, SYBR green real-time RT-PCR is still a sensitive and consistent method for ORSV detection in orchids. With additional controls, this method could prove to be the ideal method for reliable detection of ORSV in commercially-grown orchids.