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Phytoplasmas pp 159-169 | Cite as

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

  • Claudio RattiEmail author
  • Stefano Minguzzi
  • Massimo Turina
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1875)

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.

Key words

Crude extract DNA RNA extraction Phytoplasmas qPCR 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Claudio Ratti
    • 1
    Email author
  • Stefano Minguzzi
    • 1
  • Massimo Turina
    • 2
  1. 1.Department of Agricultural and Food Sciences (DISTAL)University of BolognaBolognaItaly
  2. 2.Institute for Sustainable Plant ProtectionNational Research Council of Italy, IPSP-CNRTurinItaly

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