Table 3.
Validation of 23S rRNA LAMP assay according to the EPPO standards
| Performance criteria | Result | Verification method |
|---|---|---|
| Analytical sensitivity (DNA) | 9–27 copies of FDp DNA (1:270 dilution of FDp DNA) | 3 experiments with 8 serial dilutions of DNA were performed. Maximum dilution of FDp DNA that was detected was 1:270. |
| Analytical sensitivity (homogenate) | 9–27 copies of FDp DNA (1:81 dilution of FDp infected homogenate) | 3 experiments with at least 6 serial dilutions of plant homogenate were performed. Maximum dilution of FDp DNA that was detected was 1:81. |
| Analytical specificity | 100% accuratea | No. of targets analysed: 65 + 2 (EY)b. No. of non-targets analysed: 123 (for details see Table 3). |
| Selectivity | There was no impact observed of different hosts, grapevine cultivars or tissues on the test results. | FD was confirmed using LAMP in 12 different grapevine cultivars, either in berries or leaf veins, and also in C. vitalba, A. glutinosa, O. ishidae and S. titanusc |
| Repeatability | High FDp conc: 100% (29 pos/29 repeats) | At least 3 replicates of DNA sample with low (8 samples with <81 copies of FDp DNA), medium (4 samples with 81–729 copies of FDp DNA) and high (5 samples with >729 copies of FDp DNA) concentration of FDp were analysed. |
| Medium FDp conc: 100% (12 pos/12 repeats) | ||
| Low FDp conc: 81% (22 pos/27 repeats) | ||
| Reproducibility | High FDp conc: 100% (3 pos/3 repeats) | 2 replicates of DNA sample with low (8 samples with <81 copies of FDp DNA), medium (2 samples with 81–729 copies of FDp DNA) and high (3 samples with >729 copies of FDp DNA) concentration of FDp were analysed. Analyses were performed on 2–9 different days, by two different operators and two different devices. |
| Medium FDp conc: 100% (2 pos/2 repeats) | ||
| Low FDp conc: 100% (8 pos/8 repeats) | ||
| Diagnostic sensitivity (DNA) | 100% | No. of targets analysed with LAMP and qPCR: 52 FDp infected samples (38 grapevine leaf vein, 8 C. vitalba, 3 A. glutinosa, 1 S. titanus and 2 O. ishidae samples) |
| Diagnostic sensitivity (homogenate) | 100% | No. of targets analysed with LAMP and qPCR: 27 FDp infected grapevine samples |
| Diagnostic specificity | 100% | No. of non-targets analysed with LAMP and qPCR: 53 FDp non-infected samples (48 grapevines, 2 C. vitalba, 1 A. glutinosa, 1 O. ishidae and 1 S. titanus) |
Conc, relative concentration of the FDp DNA estimated from the dilution series.
a
Calculated from the ratio between the number of correct results and the number of all results.
b
23S rRNA LAMP is specific to 16SrV phytoplasmas including EY-phytoplasma. Detailed description of the validation is in the text.
c
Clematis vitalba, Alnus glutinosa, Orientus ishidae, Scaphoideus titanus.