A fast one-step reverse transcription and polymerase chain reaction (RT-PCR) amplification procedure providing highly specific complementary DNA from plant virus RNA.
Reverse transcription and polymerase chain reaction (RT-PCR) are being used increasingly for detection and typing RNA viruses. For this purpose, metal block thermal cyclers (MBTC) are considered to provide higher DNA yield, whereas air thermal cyclers (ATC) allow PCR amplification in a much shorter time. A fast ATC protocol (0 s denaturation, 0 s annealing, and 4-8 s elongation) was developed to amplify genomic segments from two RNA viruses, which allowed increasing the number of cycles without a parallel increase of non-specific DNA fragments. Under these conditions, 80-90 cycles with the ATC provided a DNA yield close to that of a standard 40-cycles MBTC protocol in about half the time. The DNA synthesised by the new procedure was highly specific and could be cloned readily.
Sambade
A
A
Instituto Valenciano de Investigaciones Agrarias, Cra. Moncada-Náquera Km. 4.5, 46113-Moncada, Valencia, Spain. asambade@ivia.es
Martín
S
S
Olmos
A
A
García
M L
ML
Cambra
M
M
Grau
O
O
Guerri
J
J
Moreno
P
P
D002957:Citrus / Q000821:virology*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D010942:Plant Viruses / Q000302:isolation & purification*; D012328:RNA Viruses / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D018076:DNA, Complementary