Comparing the effectiveness of real-time PCRs to simultaneously detect and identify viable Globodera pallida and G. rostochiensis.

Abstract

Recently, the Commission Implementation Regulation (CIR) 2022/1192 has been published in Europe, prescribing regulations on eradication measures for quarantine potato cyst nematodes including mandated assays for species identification. The CIR 2022/1192 provides a derogation on the use of Beniers et al. (2014) real-time PCR as the validation process was still ongoing. In this study, the validation of a modified version of the Beniers real-time PCR was carried out, following EPPO Standard PM 7/98. Furthermore, the performance was compared to the conventional PCR of Bulman & Marshall (1997) and the Gamel et al. (2017) real-time PCR, both DNA based, which are mandated by CIR 2022/1192. Additionally, the effect of cyst viability was compared for the two real-time PCR assays. The modified Beniers real-time PCR met all validation performance criteria, with analytical sensitivity of 1 living egg for both target Globodera pallida and G. rostochiensis and scoring 100% for analytical specificity, reproducibility, robustness and diagnostic specificity. The viability assays showed that the DNA based Gamel real-time PCR provided positive results for non-viable cysts in contrast to the modified RNA based Beniers real-time PCR. Based on the results of this study, the modified Beniers qualifies for the detection and identification of Globodera pallida and G. rostochiensis and performs at least equally well as the other two PCR assays. For development and improvement of diagnostic protocols, it is recommended that competent authorities should be allowed to use recognized diagnostic protocols when in-house validated instead of only mandated tests as prescribed by CIR 2022/1192.