VALIDACIJA METODE duRT-qPCR ZA DOLOČANJE VIROIDA RAZPOKANOSTI SKORJE AGRUMOV (CBCVd) NA HMELJU Z UPORABO mRNA1192 INTERNE KONTROLE.

In order to expand the scope of accreditation in the Diagnostic Laboratory for Plant Protection (DL) at the Slovenian Institute of Hop Research and Brewing (IHPS) for the diagnostic method "duRT-qPCR for detection of CBCVd and internal control mRNA1192" (MDL 12), we performed its partial validation and analysed the analytical sensitivity, selectivity, repeatability and reproducibility. Analyses were performed using two sets of reagents (SensiFast and AgPATH). For analytical sensitivity, the limit of detection (LOD) was set at 5 pg and the limit of quantification (LOQ) at 50 pg RNA. As part of sensitivity and selectivity testing, we compared different hop cultivars, infection methods, viroid combinations and two nucleic acid isolation methods. In all tests we obtained accurate results. Repeatability and reproducibility were tested with four analysts over three days, with two sets of reagents, on set of 30 samples. We performed a total of 12 analyses and tested 360 samples, and determined them with 100 % accuracy. In the case of SensiFast kit, we obtained lower Cq values and chose it as the optimal choice for duRT-qPCR. In addition, with the MDL 12 method, we also performed an analysis of hop samples and RNA included in inter-laboratory testing and were 100 % successful. Through validation, we proved that we provide reliable results and that the method is suitable for accreditation. The paper presents the validation of the diagnostic method, which brings new findings in the field of RT-qPCR method implementation. [ABSTRACT FROM AUTHOR]