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Table 3 Comparison of different DNA extraction and LAMP protocols for Xylella fastidiosa, Ceratocystis platani and Phytophthora ramorum detection
| Protocol | This study | Tomlinson et al. (2007) | Harper et al. (2010) | ||
|---|---|---|---|---|---|
| DNA extraction | |||||
| Target pathogen | Xylella fastidiosa, Ceratocystis platani, Phytophthora ramorum | Phytophthora ramorum | Xylella fastidiosa | ||
| Commercial kit | Plant Material Lysis Kit (OptiGene) | EZNA Plant DNA Kit (Omega Bio-tek) | QuickPick Plant DNA kit (Bio-Nobile) | Invimag Plant DNA Mini Kit (Invitek) | DNeasy Plant Minikit (Qiagen) |
| Use | Field | Laboratory | Field | Laboratory | Laboratory |
| Sample requirement | Fresh plant tissue (80–100 mg) | Fresh plant tissue (80–100 mg) | Fresh plant tissue (15–25 mg) | Lyophilized petiole (200 mg) | Fresh plant tissue (200 mg) |
| Advantages | Rapid and simple protocol with few reagents and steps; no laboratory instruments are required | Protocol kit with spin columns and buffer supplied | Processing up to 24 samples in parallel | Simplified sample processing | Grounding with beads; kit with spin column and buffer supplied |
| Disadvantage | Difficult for large number of samples | Required laboratories facilities for grinding and DNA extraction | Extremely basic equipment is needed | Required laboratories facilities for grinding and for DNA extraction | Required laboratories facilities for grinding and for DNA extraction |
| Time per sample | 5 min | 1 h | 40–50 min | > 30 min | 1 h |
| Isothermal DNA amplification | |||||
| Instrument | Genie II (OptiGene) | Smart Cycler (Cepheid) | ABI 9700 Thermocycler (Applied Biosystems) | ||
| Use | Field | Laboratory | Laboratory | ||
| Sensitivity (LOD) | P. ramorum (0.128 pg) | P. ramorum (10 pg) | – | ||
| X. fastidiosa (0.02 pg) | – | X. fastidiosa (1.4 pg) | |||
| C. platani (0.02 pg) | – | – | |||
| Specificity | P. ramorum (high specific; P. lateralis) | P. ramorum (high specific; P. lateralis) | – | ||
| X. fastidiosa (very high specific) | – | X. fastidiosa (very high specific) | |||
| C. platani (high specific; C. fimbriata) | – | – | |||
| Advantages | Rapid detection results; amplification and detection reaction is carried out in the same instrument (16 sample per run) | High number of samples to be processed | High number of samples to be processed | ||
| Disadvantage | Strip tubes with amplification mix need to be prepared before in laboratory | Additional steps to visualize amplified products (electrophoresis gel, colorimetric detection, fluorescent dye) | Electrophoresis gel to visualize amplified products | ||
| Time per sample | 30 min | > 1 h | > 1 h | ||
