Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid

Abstract

Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimized for the simultaneous detection of CEVd and HSVd. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Two different RNA extraction methods and one rapid crude template preparation procedure were compared in terms of extraction purity and efficiency for PCR applications. Extraction method Q included a commercially available kit, whereas method C was a modified chloroform-phase extraction in house protocol. Procedure S involved blotting the sap extract on a positively charged nylon membrane and elution. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional RT-PCR tests, the overall Dse and Dsp were lower and estimated at 94 and 95% for CEVd, and 97 and 98% for HSVd, respectively. In a direct comparison, the developed assay presented 1000-fold more analytical sensitivity. Spectrophotometric results showed that RNA extraction methods Q and C, yielded the purest RNA, and gave the lowest mean Ct values. Alternative template preparation method S resulted in Ct values statistically similar to those obtained with methods Q to C when tested by RT-TaqMan PCR. The developed assay, using crude template preparation S, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and certification schemes.

Keywords: Assay performance; CEVd; HSVd; Sensitivity; Specificity; TaqMan.