|HAJERI, SUBHAS - Central California Tristeza Eradication Agency|
|VIDALAKIS, GEORGIOS - University Of California|
|Yokomi, Raymond - Ray|
Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: 12/16/2020
Publication Date: 12/4/2021
Citation: Hajeri, S., Vidalakis, G., Yokomi, R.K. 2021. Detection of viroids using RT-qPCR. In: Rao, A.L.N., Lavagi-Craddock, I., Vidalakis, G., editors. Viroids. Methods in Molecular Biology, vol 2316. New York, NY: Humana. p. 153-162. https://doi.org/10.1007/978-1-0716-1464-8_14.
Technical Abstract: Viroids are the smallest infectious pathogens known, nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has revolutioned the way the viroids are detected. The method involves using sequence specific primers that annealed to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR. Alternatively, with One-Step RT-qPCR System, viroids can be detected in a single tube, which requires less hands-on time to set up an assay than standard Two-Step RT-qPCR.