Detection of Strawberry necrotic shock virus using conventional and TaqMan(®) quantitative RT-PCR.
Graft-indexing of an advanced selection from the University of Florida strawberry breeding program produced virus-like symptoms on Fragaria vesca. However; RT-PCR testing of the material did not detect the presence of any of 16 strawberry virus species or members of virus groups for which strawberries are routinely indexed. Large scale sequencing of the material revealed the presence of an isolate of Strawberry necrotic shock virus. The nucleotide sequence of this isolate from Florida shows a significant number of base changes in the annealing sites of the primers compared to the primers currently in use for the detection of SNSV thereby explaining the most probable reason for the inability to detect the virus in the original screening. RT-PCR and Taqman(®) qPCR assays were developed based on conserved virus sequences identified in this isolate from Florida and other sequences for SNSV currently present in GenBank. The two assays were applied successfully on multiple samples collected from several areas across the United States as well as isolates from around the world. Comparison between the RT-PCR and the qPCR assays revealed that the qPCR assay is at least 100 times more sensitive than conventional PCR.
Department of Plant Pathology, Division of Agriculture, University of Arkansas System, Fayetteville, AR 72701, United States.
Thanuja
T
Thekke Veetil
Department of Plant Pathology, Division of Agriculture, University of Arkansas System, Fayetteville, AR 72701, United States.
Thien
T
Ho
Gulf Coast Research and Education Center, IFAS, University of Florida, Wimauma, FL 33598, United States.
Catalina
C
Moyer
Gulf Coast Research and Education Center, IFAS, University of Florida, Wimauma, FL 33598, United States.
Vance M
VM
Whitaker
Department of Plant Pathology, Division of Agriculture, University of Arkansas System, Fayetteville, AR 72701, United States. Electronic address: itzaneta@uark.edu.
Ioannis E
IE
Tzanetakis
D017931:DNA Primers; D031985:Fragaria / Q000821:virology*; D019179:Ilarvirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D057230:Limit of Detection; D015345:Oligonucleotide Probes; D010935:Plant Diseases / Q000821:virology*; D012367:RNA, Viral / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012367:RNA, Viral
Strawberry necrotic shock virus; Taqman qPCR; Virus detection