One-step multiplex RT-PCR for simultaneous detection of four pome tree viroids

Abstract

Apple scar skin viroid (ASSVd), Apple dimple fruit viroid (ADFVd), Apple fruit crinkle viroid (AFCVd), and Pear blister canker viroid (PBCVd) infect pome fruit trees. These viroids are important quarantine pathogens for the international movement of pome germplasm. A single-step multiplex reverse transcription polymerase chain reaction assay (mRT-PCR) was developed for the simultaneous detection of these viroids. Four pairs of primers specific for each of the four viroids were used to amplify PCR products of different sizes that can be resolved by agarose gel electrophoresis. Amplification of a plant 18S rRNA was included in the assay as an internal control. Amplicons of 371 bp (AFCVd), 270 bp (ADFVd), 186 bp (ASSVd), 120 bp (PBCVd), and 844 bp (18S rRNA) were obtained in both uniplex and mRT-PCR assays. The identities of the amplification products were confirmed by sequencing. The specificities and limits of detection for all four viroids by uniplex and mRT-PCR assays were comparable. The assay was further validated using samples from pome trees inoculated with all four viroids, as well as field samples from commercial orchards in Colorado. All four viroids were detected from inoculated pear trees and up to three viroids were detected from inoculated apple trees. This is a simple, rapid and cost-effective technique to detect these four viroids in fruit trees. The procedure is especially applicable to certification and quarantine programs, where numerous samples must be tested for all four viroids.Keywords Apscaviroid . Apple dimple fruit viroid . Apple fruit crinkle viroid . Apple scar skin viroid . Multiplex RT-PCR . Pear blister canker viroid

Development of a duplex one-step RT-qPCR assay for the simultaneous detection of Apple scar skin viroid and plant RNA internal control

“…Detection methods based on biological indexing by grafting onto woody indicators is expensive for importers, indicators plants may take up to 5 years to develop symptoms and the results can be greatly affected by environmental conditions (Desvignes et al, 1999). For optimum symptom expression, woody indexing needs to be carried out in the field, which therefore increases the chances of accidental spread of any pathogens present (Lin et al, 2012). Furthermore, biological indexing is resource intensive, especially when dealing with large numbers of samples.…”

On the early identification and characterization of pear blister canker viroid, apple dimple fruit viroid, peach latent mosaic viroid and chrysanthemum chlorotic mottle viroid

“…A20 and, moreover, helped to discard the involvement of PBCVd in other pear disorders such as bark split, bark necrosis and bud drop. In the following decades, new detection techniques were developed that included tissue-printing hybridization (Lolic et al, 2007), RT-PCR standard protocols (Faggioli and Ragozzino, 2002;Hassan et al, 2006), one-step multiplex RT-PCR (Ragozzino et al, 2004;Lin et al, 2012), standard and multiplex RT-PCR-probe capture hybridizations (Shamloul et al, 2002) and, more recently, quantitative RT-PCR (Malandraki et al, 2015;Beaver et al, 2022). The exploitation of Fig.…”

Diagnostic techniques for viroids

“…Simultaneous detection lessens costs, labour and time but, at the same time, the amount of amplified DNA is lower than with single RT-PCR (Ito et al, 2002a;Narayanasamy, 2011a). Nevertheless, multiplex RT-PCR reactions, such as fluorescent mRT-PCR (Di Serio et al, 2002), mRT-PCR-ELISA (Shamloul et al, 2002) and regular mRT-PCR (Nie & Singh, 2000;Ito et al, 2002a;Ragozzino et al, 2004;Hataya, 2009;Wang et al, 2009;Matsushita et al, 2010;Hajizadeh et al, 2012;Lin et al, 2012;Lu et al, 2012;Song et al, 2013;Gambino et al, 2014;Kumar et al, 2014;Olivier et al, 2014) are increasingly being used.…”