Tube-Capture (TC) RT-PCR and Multiplex RT-PCR for Diagnosis and Characterization of Viruses in Fruit Trees.

Abstract

Diagnosis of fruit tree viruses has been challenging for a long time as viral titer is often low and unevenly distributed among different tissues and branches of fruit trees. It is necessary to develop effective and reliable detection systems to identify viral pathogens in fruit trees. In this chapter, I describe RT-PCR and its derivatives tube capture-based reverse-transcription PCR (TC-RT-PCR) and multiplex RT-PCR assays for detection and identification of latent viruses in apple and pear trees. Classical RT-PCR is composed of two steps including transcription of viral RNA using extracted total RNA and PCR amplification of viral cDNA. TC-RT-PCR includes a TC step to capture particles and nucleic acid mixtures from crude plant tissue extracts as template directly for the first single-strand DNA (cDNA) synthesis, followed by PCR to amplify the viral cDNA fragment for viral identification. The cDNA derived from total RNAs can also be used for a one-step multiplex PCR to simultaneously detect several viruses in a given sample. As perennial fruit trees are usually coinfected by several viruses in orchards, multiplex RT-PCR can save time and lower labor and material costs for viral detection. These nucleic acid-based methods are sensitive and may be adapted for detection and identification of diverse viruses from different tissue materials of fruit trees.