A fast one-step reverse transcription and polymerase chain reaction (RT-PCR) amplification procedure providing highly specific complementary DNA fromplant 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, w hereas air thermal cyclers (ATC) allow PCR amplification in a much shorter time. A fast ATC protocol (0 a denaturation, 0 s annealing, and 4-8 s elong ation) was developed to amplify genomic segments from two RNA viruses, whic h allowed increasing the number of cycles without a parallel increase of no n-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 i n about half the time. The DNA synthesised by the new procedure was highly specific and could be cloned readily. (C) 2000 Elsevier Science B.V. All ri ghts reserved.