Optimising fast polymerases for the rapid detection of the Ralstonia solanacearum species complex by real-time PCR (EPPO Conference on Diagnostics of Plant Pests, 2026)

The Ralstonia solanacearum species complex (RSSC) poses a significant threat to plant health, necessitating rapid and reliable detection methods. Conventional real-time PCR (qPCR) workflows can take up to 1 hour and 40 minutes, which limits turnaround time in routine diagnostics and trade contexts. Although fast polymerases offer potential time savings, their performance must first be evaluated in plant matrices.

This study assessed whether fast qPCR polymerases could reduce analysis time while maintaining the ability to detect RSSC in potato extracts. Two assays were evaluated using both pure DNA and plant extract matrices across nine commercially available mastermixes.

Amplification of positive controls was generally consistent across master mixes, whereas performance in plant extracts varied, indicating the presence of matrix-dependent inhibitory effects. Several fast polymerases demonstrated comparable performance to the standard protocol, while significantly reducing analysis time. In particular, the Fast Universal PCR Master Mix and the Premix Ex Taq showed reliable performance with the Weller_B2 assay. Conversely, some formulations exhibited reduced specificity, non-specific amplification or decreased reliability, and were therefore excluded from further consideration.

Reduced efficiency due to matrix inhibition was observed for the Vreeburg assay, which supports the replacement and validation of the test with NYTor (Vreeburg et al., 2018). Including Ralstonia pseudosolanacearum within RSSC further highlights the importance of assays that can detect a broad range of taxa within the complex.

These results demonstrate that, while fast polymerases can improve diagnostic throughput, they require careful, assay- and matrix-specific validation. The study provides practical guidance for selecting qPCR chemistries in routine plant diagnostics.

This poster was presented at the EPPO conference, 'Diagnostics of Plant Pests: Recent Developments and Future Trends', which took place in Vienna, Austria, from 22 to 24 April 2026.

This work builds on a previous version that was presented at a national conference and is available at https://zenodo.org/records/18757491.

Disclaimer: results reflect the specific assays, sample matrices and reagent batches evaluated in this study and may vary under different conditions.