Inter-laboratory comparison of four RT-PCR based methods for the generic detection of pospiviroids in tomato leaves and seeds

Four pospiviroid detection methods consisting of a pair of RT-PCR (ANSES 1-2), a pair of real time RT-PCR (Botermans 1-2) and two single RT-PCR methods (Luigi and Olivier) were evaluated for their relative accuracy, diagnostic sensitivity, diagnostic specificity, analytical sensitivity and reproducibility through a comparison in eight laboratories. All detection methods were tested on 13 tomato leaf and nine tomato seed samples as well as on 100-fold dilutions of the corresponding RNA extracts. Two different RNA extraction kits and three combinations of RNA extraction and RT-PCR kits were assessed. According to the statistical analysis of the results, ANSES 1-2 method provided the best performance criteria whatever the matrix consisted of, while the relative accuracies of Botermans 1-2 and Olivier methods were not significantly different from ANSES 1-2 for seed and leaf samples respectively. The results also showed that the relative accuracy of the Luigi and Olivier methods significantly increased when primer concentrations were raised to 1 μM, the latter method giving the best relative accuracy of the test in these conditions. No statistical differences were observed between accuracies obtained for the two extraction kits or the three combinations of extraction and RT-PCR kits used. ANSES 1-2 method is proposed as international standard for the generic detection of pospiviroids on tomato leaves and seeds.