A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification

Area of Science:

• Plant pathology
• Molecular diagnostics
• Phytoplasmology

Background:

• Traditional phytoplasma detection relies on genomic DNA extraction and amplification.
• This process is time-consuming and costly, especially for large-scale monitoring programs.

Purpose of the Study:

• To develop a faster, more cost-effective phytoplasma diagnostic protocol.
• To maintain high sensitivity and specificity in phytoplasma detection.

Main Methods:

• A novel approach avoiding nucleic acid purification.
• Exploiting amplification of abundant phytoplasma ribosomal RNA (rRNA).
• Utilizing quantitative PCR (qPCR), reverse transcription qPCR (RT-qPCR), digital droplet PCR (ddPCR), and digital droplet RT-PCR (ddRT-PCR) with TaqMan chemistry.

Main Results:

• The new protocol significantly reduces analysis time and costs.
• Sensitivity and specificity remain unaffected compared to traditional methods.
• The method is effective for detecting and quantifying multiple phytoplasma species.

Conclusions:

• This rRNA-based diagnostic approach offers an efficient alternative for phytoplasma detection.
• It is particularly valuable for large-scale certification programs and monitoring quarantine pathogens.
• The protocol supports the detection of Candidatus Phytoplasma mali, Ca. P. pyri, Ca. P. prunorum, Ca. P. vitis, and Ca. P. solani.