Comparative procedures for sample processing and quantitative PCR detection of grapevine viruses

Fatima Osman, Tammi Olineka, Emir Hodzic, Deborah Golino, Adib Rowhani

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

In this study different instruments and methods used for tissue homogenization, RNA extraction and quantitative PCR (qPCR) based detection of grapevine RNA viruses were evaluated. Semi-automated and automated homogenization techniques were compared to process samples from grapevine petioles and cambial tissue. Four different high throughput automated nucleic acid extraction platforms were compared with the RNeasy plant extraction kit for their capacity and efficiency of extracting viral RNA from grapevine infected tissues. The RNA prepared from each extraction platform was then used as template for a comparative analysis of qPCR by One Step RT-qPCR, Two Step RT-qPCR and low density array (LDA) detection. This study showed that a thorough homogenization of grapevine tissues using the Tissue Lyser as well as DNase digestion of the purified RNA prior to cDNA synthesis improved the virus detection and yielded the lowest quantitation cycle (Cq) values in RT-qPCR. Comparison of different RNA extraction methods showed that methods implementing the magnetic bead-based technology were superior to other methods used. Comparing different qPCR detection methods, One Step RT-qPCR showed the lowest Cq values for the same sample tested compared to Two Step RT-qPCR and LDA.

Original languageEnglish (US)
Pages (from-to)303-310
Number of pages8
JournalJournal of Virological Methods
Volume179
Issue number2
DOIs
StatePublished - Feb 2012

Keywords

  • Grapevine
  • LDA
  • RNA extraction
  • RT-qPCR
  • Virus detection

ASJC Scopus subject areas

  • Virology

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