Submitted to: Journal of Virological Methods
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/30/2007
Publication Date: 1/1/2008
Publication URL: www.sciencedirect.com
Citation: Saponari, M., Keremane, M.L., Yokomi, R.K. 2008. Quantitative Detection of Citrus tristeza virus (CTV) in Citrus and Aphids by Real-time Reverse Transcription-PCR (TaqMan®). Journal of Virological Methods. 147:43-53. Interpretive Summary: A quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay for rapid detection of Citrus tristeza virus (CTV) was developed and tested successfully on a wide array of biologically diverse CTV isolates collected from around the world. The qPCR method uses fluorescent molecules to measure the PCR directly while amplification is taking place. This molecular assay is faster and more sensitive than serology or conventional RT-PCR. Concentrations of less than 2 fentograms could be detected from infected citrus tissue while the amount of virus in aphids was estimated to range from 12,000 to 13,000,000 copies of CTV RNAs. In summary, the real time RT-PCR assay developed is a valuable new tool for CTV detection and quantification.
Technical Abstract: Routine detection of Citrus tristeza virus (CTV) is by enzyme-linked immunosorbent (ELISA) and direct tissue blot immunoassays. Reverse transcription (RT) polymerase chain reaction (PCR) has also been developed for CTV detection which is more sensitive than serology. We developed a quantitative and multiplex real time RT-PCR assay for simultaneous detection of CTV and plant mRNA as an internal control. The technique successfully detected different CTV strains in plants and aphids. A dilution series using an in vitro synthesized full length CTV-CP transcript containing the target sequence demonstrated that the assay was effective for quantification of the viral template in infected plants and in single aphids. CTV detection was compared from different plant tissues and for different RNA isolation methods from aphids. Less than 2 fg were consistently detected when RNA transcripts were diluted in extracts from healthy plants while RNA copies carried by single aphids were estimated to be between 12,000 and 13,000,000. The assay was more sensitive and less time consuming than either ELISA or traditional RT-PCR. The incorporation of the RNA-specific internal control increased the reliability of the results. In summary, the real time RT-PCR assay developed is a valuable new tool for CTV detection and titer quantification.