CONTINUOUS RT-PCR USING AMV-RT AND TAQ DNA-POLYMERASE - CHARACTERIZATION AND COMPARISON TO UNCOUPLED PROCEDURES

A continuous reverse transcription polymerase chain reaction (RT-PCR) procedure was designed,vith all reaction components included in a sing le tube prior to thermal cycling. This procedure was compared to uncou pled RT-PCR procedures wherein the addition of reagents was separated. In the latter in particular; conditions for reverse-primer annealing and cDNA synthesis were investigated. The two RT-PCR approaches were c ompared in the detection of singly spliced and multiply spliced human immunodeficiency virus type I (HIV-I) mRNAs. The avian myeloblastosis virus reverse transcriptase and Tag DNA Polymerase were used in the co ntinuous procedure under the compromised condition wherein the two enz ymes were active in the same buffer Reverse transcription was carried out at an elevated temperature of 50 degrees C to overcome problems of mRNA secondary structure that could inhibit the reaction. The continu ous procedure was found to be as specific and efficient as the best un coupled procedure. The procedure was shown to be reliable and to have the sensitivity to detect one HTLV-IIIB-infected H9 cell in a million uninfected H9 cells.