Simultaneous Detection of the Three Ilarviruses Affecting Stone Fruit Trees by Nonisotopic Molecular Hybridization and Multiplex Reverse-Transcription Polymerase Chain Reaction

Abstract

The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

Epidemiología y variabilidad patogénica del virus del mosaico del pepino dulce (Pepino mosaic virus). Nuevas enfermedades asociadas a su presencia (torrao o cribado)

“…Although the multiplex RT-PCR procedure presented herein contains a cocktail of six different primers, no differences in terms of detection limit were observed when a serially-diluted double-infected sample was analyzed by either the multiplex (containing a cocktail of six primers) or the single (containing two primers) RT-PCR. When the similarly diluted samples were analyzed by ELISA or dot-blot hybridization, the detection limit of the multiplex reaction was 3125 (5 5 ) times higher, a difference that is in the range of previously reported results (Sánchez-Navarro et al, 1998;Saade et al, 2000).…”

“…El método resultó ser altamente específico incluso en infecciones múltiples, presentando a su vez un límite de detección 3125 veces mayor que el ELISA o la hibridación molecular, valor que se encuentra dentro del rango descrito para estas técnicas (Saade et al, 2000;Sánchez-Navarro et al, 1998;Sánchez-Navarro et al, 2006). El análisis de muestras de campo reveló la presencia de un gran porcentaje de muestras con infecciones mixtas de aislados pertenecientes a los genotipos EU con CH2 o CH1, representativos de dos regiones geográficas diferentes (Murcia y las Islas Canarias, respectivamente).…”

Standardization of duplex RT-PCR for simultaneous detection of Peanut bud necrosis virus and Tobacco streak virus naturally infecting peanut (Arachis hypogeae L.)

“…Simultaneous detection of Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV) by multiplex RT-PCR in woody hosts. The Multiplex RT-PCR could be a useful for indexing these three ilarviruses, it reduces time and cost for the analysis of plants in certification programs [27] . A RT-PCR assay that can detect and identify five seed borne viruses of subterranean clover was reported earlier [28] .…”

Ilarviruses and the Importance of Certified Elite Planting Material in Apple Production System - An Overview

“…Saade et al, (2000) developed nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies that could detect ApMV, PNRSV and ADV in 5 stone fruits simultaneously. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers.…”