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