Comparison of TaqMan and Epoch Dark Quenchers during real-time reverse transcription PCR.
Several biotechnology companies have recently introduced novel quencher fluors for use with dual-labeled fluorogenic hydrolysis probes. The Epoch Dark Quencher trade mark fluorochrome consists of a non-fluorescent moiety capable of absorption at higher wavelengths (400-650 nm). The aim of this study was to: (1) evaluate the feasibility of using Epoch Dark Quencher fluorochromes in real-time PCR pathogen detection assays that were previously optimized with TaqMan (TAMRA) quenching fluors, and (2) compare the sensitivity based on cycle threshold (CT) between probes containing either TaqMan or Epoch Dark Quencher fluors. Our data indicate Epoch Dark Quencher probes can be used in place of TaqMan probes and their performance was not better than traditional TaqMan (TAMRA) quenchers. Marginal differences observed between quenching fluorochromes may arise from concentration differences during probe synthesis.
Daum
Luke T
LT
Brooks City-Base San Antonio, TX, USA. luke.daum@brooks.af.mil
Ye
Keying
K
Chambers
James P
JP
Santiago
Jose
J
Hickman
John R
JR
Barnes
William J
WJ
Kruzelock
Russell P
RP
Atchley
Daniel H
DH
D005456:Fluorescent Dyes / Q000378:metabolism; D009980:Influenza A virus / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism*
D005456:Fluorescent Dyes; D012367:RNA, Viral; D019914:Taq Polymerase