Development of a quantitative real-time RT-PCR (qRT-PCR) for the detection of hop stunt viroid

Abstract

Hop stunt viroid (HSVd) is a worldwide plant pathogen that infects a wide range of hosts. In some cases different molecular variants are associated with particular disease symptoms, e.g. the cachexia isolate on citrus. A new method for HSVd detection has been developed by designing a new primer pair and a probe for quantitative real-time RT-PCR (qRT-PCR) assay that is based on TaqMan™ chemistry. The primers and the probe were manually designed in order to detect different HSVd isolates from different hosts, including citrus cachexia variant. The protocol was then tested for its sensitivity and specificity. Specifically, sensitivity was evaluated with dilution series of in vitro transcripts from cloned whole HSVd genome. Dilutions were used as a standard for quantification of viroidal template in infected samples. Sensitivity was also tested by comparing the amplification results in quantitative and qualitative (end point) RT-PCR, obtained with serially diluted total RNA, extracted with two different methods. The qRT-PCR was evaluated for its specificity using healthy and infected samples from peach, apricot, plum, pear, citrus (infected with cachexia and non-cachexia variants), almond and grapevine and non-target controls (Apple scar skin viroid, Apple dimple fruit viroid, Peach latent mosaic viroid, Pear blister canker viroid and Potato spindle tuber viroid). The assay showed very high sensitivity and specificity. When the developed assay was applied to detect HSVd variants in different hosts that were previously tested, it was in complete agreement with previous test which confirmed its efficiency and accuracy. Moreover, the developed assay has the advantages of increased sensitivity of viroid detection as well as its quantification.