High throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods.
The development and application of a novel, sensitive TaqMan fluorescent probe-based product enhanced RT test (F-PERT) for the detection of retrovirus are described. The assay allows discrimination between the amplification signals generated by genuine positive signals that result from retroviral RT activity and the RT-like activity from DNA polymerases. The RT-like activity from DNA polymerases was suppressed by the addition of activated calf-thymus DNA with no reduction in the RT activity. A linear relationship between threshold cycle (C(T)) and the number of virus particles was demonstrated, allowing quantification of retroviruses in unknown samples. The F-PERT assay was able to detect a wide range of retroviral RT activities, including that from porcine endogenous retrovirus (PoERV), murine leukaemia virus (MLV), simian foamy virus (SFV), simian immunodeficiency virus (SIVmac) and squirrel monkey retrovirus (SMRV). The detection limit of SMRV, MLV and PoERV was approximately 100 virion particles and the test was able to detect at least 10(2) molecules of purified RT enzyme. RT activity was not detected in cellular lysates and supernatants from MRC-5, BT, VERO, or Raji cells, whereas RT activity was detected in C1271, Mus dunni, K-Balb, BHK-21, CHO-K1, SP2/0-Ag14 and NSO cell supernatants. RT activity was also detected in the Spodoptera cell line Sf9.
Lovatt
A
A
Q-One Biotech Ltd, Glasgow, UK.
Black
J
J
Galbraith
D
D
Doherty
I
I
Moran
M W
MW
Shepherd
A J
AJ
Griffen
A
A
Bailey
A
A
Wilson
N
N
Smith
K T
KT
D000818:Animals; D002384:Catalysis / Q000187:drug effects; D002413:Cations, Divalent / Q000494:pharmacology; D002460:Cell Line; D017077:Culture Media, Conditioned; D004247:DNA / Q000494:pharmacology; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005189:False Positive Reactions; D005456:Fluorescent Dyes / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D012190:Retroviridae / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014764:Viral Proteins / Q000032:analysis
D002413:Cations, Divalent; D017077:Culture Media, Conditioned; D005456:Fluorescent Dyes; D019384:Nucleic Acid Synthesis Inhibitors; D014764:Viral Proteins; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase