title: One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.
abstract: A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys.
authors.affiliation: Benaki Phytopathological Institute, Department of Phytopathology, Laboratory of Virology, St. Delta 8, 14561 Athens, Greece.
authors.forename: Ioanna
authors.initials: I
authors.lastname: Malandraki
authors.affiliation: Benaki Phytopathological Institute, Department of Phytopathology, Laboratory of Virology, St. Delta 8, 14561 Athens, Greece.
authors.forename: Christina
authors.initials: C
authors.lastname: Varveri
authors.affiliation: Plant Protection Centre, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, 46113 Valencia, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Olmos
authors.affiliation: Benaki Phytopathological Institute, Department of Phytopathology, Laboratory of Virology, St. Delta 8, 14561 Athens, Greece. Electronic address: N.Vassilakos@bpi.gr.
authors.forename: Nikon
authors.initials: N
authors.lastname: Vassilakos
mesh_terms: D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D045582:Phytoplasma / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D027824:Rosaceae / Q000382:microbiology* / Q000821:virology*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014197:Trees / Q000382:microbiology / Q000821:virology; D014772:Viroids / Q000235:genetics / Q000302:isolation & purification*
keywords: Apple scar skin viroid; MGB probes; Pear blister canker viroid; Real-time PCR; ‘Candidatus Phytoplasma mali’; ‘Candidatus Phytoplasma pyri’