Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR

Ioanna Malandraki; Despoina Beris; Ioannis Isaioglou; Antonio Olmos; Christina Varveri; Nikon Vassilakos

doi:10.1371/journal.pone.0180877

Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR

PLoS One. 2017 Jul 27;12(7):e0180877. doi: 10.1371/journal.pone.0180877. eCollection 2017.

Authors

Ioanna Malandraki¹, Despoina Beris¹, Ioannis Isaioglou¹, Antonio Olmos², Christina Varveri¹, Nikon Vassilakos¹

Affiliations

¹ Benaki Phytopathological Institute, Department of Phytopathology, Laboratory of Virology, Athens, Greece.
² Plant Protection and Biotechnology Centre, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, Spain.

Abstract

A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10-4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.

MeSH terms

Fruit / virology*
Malus / virology*
Plant Viruses / physiology*
Pyrus / virology*
RNA, Plant / isolation & purification
Real-Time Polymerase Chain Reaction / methods*
Reference Standards
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction / methods*
Trees / virology*

Substances

RNA, Plant

Grants and funding

This work was supported by: 1. Hellenic Greek Secretariat of Research and Technology Project ‘09SYN-22-638’; 2. FP7-REGPOT-2008-1 project ‘BPI-PlantHeal 230010’ (Partially). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.