A simplified method for obtaining plant viral RNA for RT-PCR.
An easy and fast procedure (named the simple-direct-tube (SDT) method) was developed for preparing plant virus RNA for cDNA synthesis. The SDT method can be completed in approximately 15min and does not require the use of antiserum, filtering or centrifugation. The procedure to grind plant tissues in phosphate-buffered saline containing Tween-20 (PBST) and to place the extract in a microfuge tube for a few minutes allow adsorption of the virus particles to the tube wall. The sap is then removed and the tube is washed with PBST before the addition of RNase-free water. This manipulation can be performed at room temperature. Using this method followed by reverse transcription-polymerase chain reaction (RT-PCR), infections by turnip mosaic virus, cucumber mosaic virus, and cucumber green mottle mosaic virus (CGMMV) were readily detected, indicating that the SDT method can be used in assays to detect different viruses. For the detection of CGMMV, it was necessary to heat the tubes before cDNA synthesis, suggesting that the immobilized CGMMV particles required disruption by heat treatment to release RNA.
Suehiro
Noriko
N
Laboratory of Plant Pathology, Faculty of Agriculture, Utsunomiya University, Mine-machi 350, Utsunomiya 321-8505, Japan.
Matsuda
Kazunori
K
Okuda
Seiichi
S
Natsuaki
Tomohide
T
D017799:Cucumovirus / Q000235:genetics / Q000302:isolation & purification; D010935:Plant Diseases / Q000821:virology; D010942:Plant Viruses / Q000235:genetics* / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017898:Tobamovirus / Q000235:genetics / Q000302:isolation & purification; D017806:Tymovirus / Q000235:genetics / Q000302:isolation & purification; D014773:Virology / Q000379:methods*
D012367:RNA, Viral