Development and inter-laboratory evaluation of real-time PCR assays for the detection of pospiviroids

Duplex realtime RT-PCR assay for the detection of Potato spindle tuber viroid (PSTVd) along with ef 1-α gene of potato

“…These assays are also comparatively less time consuming and does not involve any hazardous chemicals. Taqman probe based realtime PCR assays for the detection pospiviroids including PSTVd have been developed and validated (Boonham et al 2004;Roenhorst et al 2005;Monger et al 2010;Botermans et al 2013). Boonham et al (2004) found that the real time RT-PCR was 1000 times more sensitive than the chemiluminescent hybridization detection of PSTVd and it could detect up to 1 ×10 -6 dilutions of infected RNA.…”

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).…”

Quantitation and localization of pospiviroids in aphids

“…For detection and quantitation of viroids in plant tissues and seeds, several PCR-based assays have been developed over the recent years (Boonham et al, 2004;Monger et al, 2010;Verhoeven et al, 2010;Botermans et al, 2013). However, these assays have not yet been validated for insect matrices.…”