Open AccessSimultaneous Detection of Nine Grapevine Viruses by Multiplex Reverse Transcription-Polymerase Chain Reaction with Coamplification of a Plant RNA as Internal Control
Author(s) -
Giorgio Gambino,
Ivana Gribaudo
Publication year - 2006
Publication title -
phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.264
H-Index - 131
eISSN - 1943-7684
pISSN - 0031-949X
DOI - 10.1094/phyto-96-1223
Subject(s) - biology , virus , rna extraction , virology , multiplex , agarose gel electrophoresis , reverse transcription polymerase chain reaction , polymerase chain reaction , reverse transcriptase , rna , plant virus , microbiology and biotechnology , dna , gene , genetics , messenger rna
A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection of nine grapevine viruses: Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Rupestris stem pitting-associated virus, Grapevine fleck virus, Grapevine leafroll-associated virus-1, -2, and -3, in combination with a plant RNA internal control used as an indicator of the effectiveness of RNA extraction and RT-PCR. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. Two plant total RNA extraction methods (silica capture and modified RNeasy method) and two RT-PCR systems (onestep and two-step) were evaluated to develop a reliable protocol for mRT-PCR. One to nine fragments specific for the viruses were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. In the two-step mRT-PCR, the detection limits were 10 -3 or 10 -4 extract dilutions, depending on the virus. Leaves, phloem from dormant cuttings, and in vitro plantlets from 103 naturally infected and healthy grapevines were analyzed. The mRT-PCR provided a reliable and rapid method for detecting grapevine viruses from a large number of samples.