Substitution of 3′-Phosphate Cap with a Carbon-Based Blocker Reduces the Possibility of Fluorescence Resonance Energy Transfer Probe Failure in Real-Time PCR Assays

2004 ◽  
Vol 50 (6) ◽  
pp. 1080-1082 ◽  
Author(s):  
Kendall W Cradic ◽  
Jason E Wells ◽  
Lindsay Allen ◽  
Kent E Kruckeberg ◽  
Ravinder J Singh ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Real Time Pcr ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Pcr Assays ◽  
Fluorescence Resonance ◽  
Carbon Based

scholarly journals Rapid Detection and Differentiation of Human Pathogenic Orthopox Viruses by a Fluorescence Resonance Energy Transfer Real-Time PCR Assay

2004 ◽  
Vol 50 (4) ◽  
pp. 702-708 ◽  
Author(s):  
Marcus Panning ◽  
Marcel Asper ◽  
Stefanie Kramme ◽  
Herbert Schmitz ◽  
Christian Drosten
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Internal Control ◽  
Real Time Pcr ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Pcr Assay ◽  
Strain Differentiation ◽  
Fluorescence Resonance

Abstract Background: The orthopox viruses that are pathogenic for humans include variola major virus (VAR), monkeypox virus (MPV), cowpox virus (CPV), and to a lesser extent, camelpox virus (CML) and vaccinia virus (VAC). PCR is a powerful tool to detect and differentiate orthopox viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of contamination, capability of strain differentiation, and use of multiplexed probes. Methods: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction. Results: Strain differentiation results showed little interassay variability (CV, 0.4–0.6%), and the test was 100-fold more sensitive than virus culture on Vero cells. Low copy numbers of DNA could be detected with ≥95% probability (235–849 genome copies/mL of plasma). Conclusions: The real-time PCR assay can detect and differentiate human pathogenic orthopox viruses. The use of an internal control qualifies the assay for high sample throughput, as is likely to be needed in situations of suspected acts of biological terrorism, e.g., use of VAR.

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