nPCR X. axonopodis pv. dieffenbachiae – 96 tests

The PCR Xad kit has been developed by Qualiplante according to Robène-Soustrade et al. (2006). This method is referred in the Appendix 4 of the PM7/23 (2) Xanthomonas axonopodis pv. dieffenbachiae, European and Mediterranean Plant Protection Organization Bulletin (2009) 39, 393-402.

Qualiplante PCR Xad kit is also referred in the French official method ANSES/LSV/MA 028 – Version 3, September 2019 and gave equivalent results compared to a home-made master mix (§5.2.2).

The PCR primers used in this Nested End-Point PCR method were selected from the SCAR sequence of Xad strain LMG695 and complemented some bases of the SCAR sequence. The detection threshold obtained with pure cultures or plant extracts (10³ CFU ml^-1) allowed detection of the pathogen from symptomless contaminated plant.

Internal validation data of the method are available on the ANSES/LSV/MA 028 – Version 3, September 2019 realized in 2008 by ANSES. The performance characteristics obtained are:

– Reproducibility: 93% tested during an interlaboratory test comprising 15 different laboratories covering 4 different samples (8 replicates per sample and by laboratory) made up of ground healthy anthurium doped or not with targets or non-targets at different levels of contamination.

– Sensitivity: 100% tested with 50 target bacterial strains in duplicate, with geographic and genetic diversity, diversity of host plants and years of isolation.

– Specificity: 96% tested with 53 bacterial strains or non-target samples in duplicate.

– Accuracy: 98% tested with 50 target bacterial strains in duplicate and 53 bacterial strains or non-target samples in duplicate.

– Limit of detection: [5.10³ – 5.10⁴] CFU. mL^-1.

– Repeatability: 95% tested with 6 levels of contamination 0 – 10² – 10³ – 10⁴ – 10⁵ -10⁶ CFU. mL^-1 x 4 different target strains representative of the diversity.