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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]

On hops, citrus bark cracking viroid (CBCVd) in combination with hop latent viroid (HLVd) causes the severe hop stunt disease. In order to limit further spread of the disease, the Diagnostic Laboratory for Plant Protection (DL) is developing a method for rapid detection of CBCVd in the field. In the research, we compared two standard methods of isolation of nucleic acids and two methods of "fast isolation" on samples of three hop varieties. The samples were analysed for the presence of CBCVd and HLVd viroids using reverse transcription PCR (RT-PCR) and duplex real time RT-PCR (duRT-qPCR). In the case of fast isolation, we tested the influence of inhibitors on the sensitivity of the reaction. Additionally, we also tested the effect of storage and stored the crude extract at -70 °C for a longer time period before analysis. Viroid CBCVd and HLVd were successfully determined in all four isolations using both methods. As expected, we detected inhibition and 100 times lower sensitivity with duRT-qPCR for fast isolation than when using standard methods. The variety of hops and storage had no effect on the results. The used protocols for fast isolation, thus represents a good potential for use in the detection of viroids in the field. Based on the results of the research, we can conclude that the determination of viroids without isolation of nucleic acids is very close to reality and is not only a dream of plant diagnosticians. [ABSTRACT FROM AUTHOR]

Intensive monoculture production of agricultural plants, climate change, global trade and the evolutionary ability of pathogens to adapt rapidly are main source of current emergences. Incurable plant diseases caused by viroids can limit crop production and quality and can result in significant losses. An important factor in preventing the spread is represented by diagnostic methods that can rapidly and reliably detect viroid infections. The nature of viroids, which are naked RNA without any coding potential, restricts its detection based on antigen-antibody recognition. Therefore, only nucleic acid detection methods can be applied. Some of them can be timeconsuming, require the use of expensive equipment and technical skills, which may be an obstacle for field detection. An alternative way is the use of isothermal tests, such as the loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Isothermal tests are simple, easy to perform, and reliable and can be perform using lateral flow devices. This technology has the advantage to be implemented in field-based scenarios because tests require a minimal sample preparation, and are performed at constant low temperature (37-- 42°C) without the use of sophisticated equipment. Here we briefly describe isothermal assays (LAMP and RPA), that are suitable for onsite detection and their potential for viroid detection. [ABSTRACT FROM AUTHOR]

In order to apply for accreditation of in-house method "PCR for detection of Verticillium nonalfalfae and Verticillium dahliae" (MDL 02) verification of method was performed. The method is part of EPPO standard PM 7/78 (2) "Verticillium nonalfalfae and V. dahliae", so it has been partially validated. In accordance with EPPO guidelines for the validation of methods PM 7/98 (4) we analyzed the results of previous research, performed a risk analysis and prepared a verification plan for the method. In the scope of verification we analyzed the analytical sensitivity, repeatability, reproducibility and the influence of nucleic acid isolation on the results. In the case of analytical sensitivity, the detection limit (LOD) was determined at 1 pg and the limit of quantification (LOQ) at 0.1 ng. The repeatability and reproducibility of the method were analyzed by comparing 5 operators, 2 kits and 2 PCR instruments on a set of 14 samples. A total of 338 samples were analyzed and determined with 98.8 % accuracy. Additional 95 samples isolated with CTAB reagent were analyzed and the negative impact of isolation on the results in the case of major deviations from protocol was confirmed. Our research confirmed that verification or validation of methods is crucial to obtain reliable results. [ABSTRACT FROM AUTHOR]