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 with all reaction components included in a single tube prior to thermal cycling. This procedure was compared to uncoupled 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 compared in the detection of singly spliced and multiply spliced human immunodeficiency virus type 1 (HIV-1) mRNs. The avian myeloblastosis virus reverse transcriptase and Taq DNA Polymerase were used in the continuous procedure under the compromised condition wherein the two enzymes 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 structures that could inhibit the reaction. The continuous procedure was found to be as specific and efficient as the best uncoupled 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.
Mallet
F
F
CNRS-BioMérieux, Ecole Normale Supérieure de Lyon, France.
Oriol
G
G
Mary
C
C
Verrier
B
B
Mandrand
B
B
D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D001483:Base Sequence; D002478:Cells, Cultured; D004259:DNA-Directed DNA Polymerase; D006678:HIV / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification; D012367:RNA, Viral / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D013997:Time Factors
D012333:RNA, Messenger; D012367:RNA, Viral; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase