A one-step reverse transcription real-time PCR assay for the detection and quantitation of Grapevine fanleaf virus.
A TaqMan(®) one-step reverse transcription real-time PCR (RT-qPCR) assay was developed for the specific detection and relative quantitation of Grapevine fanleaf virus (GFLV), the causal agent of grapevine fanleaf degeneration disease. The assay was targeted to a conservative region located in the 2A(HP) gene of the GFLV RNA2 molecule. The assay specificity was evaluated on GFLV isolates from a wide range of geographical regions and on other viruses infecting grapevines. The sensitivity of the developed assay for GFLV detection was approximately 1000-fold higher than the sensitivity of the conventional ELISA. Concentrations as low as 10 genome copies of GFLV per reaction were reliably detected using RT-qPCR. The new method offers a fast, reliable, specific and sensitive identification test for GFLV that is easily applicable for high-throughput diagnosis of GFLV in different types of grapevine material, including dormant phloem scrapings. The quantitative nature of the assay was evaluated by monitoring the seasonal variation of the amount of GFLV present in the plant phloem.
Cepin
Urška
U
Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, Ljubljana, Slovenia. urska.cepin@nib.si
Gutiérrez-Aguirre
Ion
I
Balažic
Leonida
L
Pompe-Novak
Maruša
M
Gruden
Kristina
K
Ravnikar
Maja
M
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D016679:Genome, Viral; D017860:Nepovirus / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D027843:Vitis / Q000821:virology*
D012367:RNA, Viral