Most of the molecular diagnostic protocols used for phytoplasmas detection are based on the purification of total nucleic acids and on the use of genomic DNA of the pathogen as the target of amplification. Here we describe a diagnostic approach that, avoiding the purification of nucleic acids and exploiting the amplification of the abundant phytoplasma ribosomal RNA molecules produced during the infectious process, allows reducing the time and the costs necessary for the analysis, without affecting sensitivity and specificity. This is useful in particular when high numbers of analyses are required, as in certification programs, to monitor phytoplasmas classified as quarantine or quality pathogens. The protocol here described can be used for the detection and quantification of Candidatus Phytoplasma mali, Ca. P. pyri, Ca. P. prunorum, Ca. P. vitis, and Ca. P. solani by qPCR, RT-qPCR, ddPCR, and ddRT-PCR techniques based on TaqMan chemistry.
Ratti, C., Minguzzi, S., Turina, M. (2019). A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification. New York, NY : Humana Press [10.1007/978-1-4939-8837-2_13].
A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification
Ratti, Claudio;
2019
Abstract
Most of the molecular diagnostic protocols used for phytoplasmas detection are based on the purification of total nucleic acids and on the use of genomic DNA of the pathogen as the target of amplification. Here we describe a diagnostic approach that, avoiding the purification of nucleic acids and exploiting the amplification of the abundant phytoplasma ribosomal RNA molecules produced during the infectious process, allows reducing the time and the costs necessary for the analysis, without affecting sensitivity and specificity. This is useful in particular when high numbers of analyses are required, as in certification programs, to monitor phytoplasmas classified as quarantine or quality pathogens. The protocol here described can be used for the detection and quantification of Candidatus Phytoplasma mali, Ca. P. pyri, Ca. P. prunorum, Ca. P. vitis, and Ca. P. solani by qPCR, RT-qPCR, ddPCR, and ddRT-PCR techniques based on TaqMan chemistry.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
