Development of quantitative real-time RT-PCR for the detection and quantification of Peach latent mosaic viroid

Abstract

Peach latent mosaic viroid (PLMVd) is a damaging pathogen for peach, its detection being of critical importance to both sanitary and certification programs worldwide. Here we report a quantitative realtime reverse transcription polymerase chain reaction assay (qRT-PCR) based on TaqMan® chemistry to improve the diagnosis of this pathogen. Critical to this approach is the design of a specific set of primers and of a probe, and the use of a suitable extraction method proving a reliable, sensitive and specific PLMVd diagnosis. More specifically, the sensitivity was evaluated using 12 ten-fold dilution series of an in vitro transcript of the entire PLMVd genome as a standard for quantification in infected samples. The assay detected up to 1 fg of target RNA. The protocol was also evaluated for its specificity using healthy peach, apricot, plum and pear controls and non-target viroids (Apple scar skin viroid, Hop stunt viroid, Pear blister canker viroid, Apple dimple fruit viroid and Potato spinder tuber viroid). None of the non-target or healthy analysed samples reacted in qRT-PCR. The efficiency and accuracy of the method was evaluated using different PLMVd peach isolates as templates, including three 'calico' variants: a strong signal was obtained from all of them. Finally, five RNA extraction methods were compared and evaluated to choose the best for detection and quantification of PLMVd by the qRT-PCR protocol.

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

“…Real-time PCR is often the method of choice for highly sensitive and fast detection and quantification of target organisms. Recently, quantitative real-time RT-PCR (RT-qPCR) has been used for detection of some viroids (Luigi and Faggioli, 2011). However, a RT-qPCR is not yet available for ASSVd.…”

Diagnostic techniques for viroids

“…RT followed by a quantitative real-time PCR (RT-qPCR) based on TaqMan and SYBR Green chemistry has been used for viroid detection (Mumford et al, 2000;Boonham et al, 2004;Rizza et al, 2009;Monger et al, 2010;Luigi & Faggioli, 2011, 2013Botermans et al, 2013;Loconsole et al, 2013). TaqMan probes enable specific hybridization between the probe and the target DNA sequence, unlike SYBR Green dye, which binds nonspecifically to double-stranded DNA.…”

“…Using SYBR Green, the reaction should be followed by melting curve analysis to avoid false positive results due to the formation of nonspecific amplicons (Mirmajlessi et al, 2015). Because TaqMan probes are more specific, the majority of assays are based on them (Mumford et al, 2000;Boonham et al, 2004;Monger et al, 2010;Luigi & Faggioli, 2011, 2013Botermans et al, 2013), while only a few use SYBR Green dye (Rizza et al, 2009;Parisi et al, 2011).…”

Rapid detection and identification of viroids in the genus Coleviroid using a universal probe

“…The simplicity and sensitivity of PCR have led this technique to be used for routine viroid diagnosis; however, false positives due to amplicon contamination, and false negatives due to the failure to generate a cDNA of suitable size during reverse transcription are not infrequent. Recently, some improvements have been made in the method to reduce the effects of contamination, such as LAMP (loop-mediated isothermal amplification) and real-time PCR procedures (Boonham et al, 2004;Boubourakas et al, 2009;Monger et al, 2010;Luigi and Faggioli, 2011); however, the cost of these methods is substantially higher. In contrast, molecular hybridization using DIG-labeled probe offers a highly dependable diagnostic method that has become attractive for routine diagnosis due to the advantages of low-cost and practicality in recent years (Herranz et al, 2005;Cohen et al, 2006;Aparicio et al, 2008;Lin et al, 2011;Zhang et al, 2012).…”