Detection of Crimean-Congo Hemorrhagic Fever Using Molecular Beacons Sawyer Ellis PBIO4500 December 2 nd , 2014
Dec 17, 2015
Detection of Crimean-Congo Hemorrhagic
Fever Using Molecular Beacons
Sawyer EllisPBIO4500
December 2nd, 2014
What is CCHF?• Virus causing hemorrhagic fever
• Ticks of Hyalomma Genus
• 10%-40% Fatality
• Africa, Balkans, Asia, Middle-East
• No Vaccine
(World Health Organization)
InfluentialPoints.com
TaqMan MBG-Probe
• TaqMan rRT-PCR Assay
• Roche Molecular Diagnostics
• Reporter gets cleaved- detection
Luke Johnson-Idaho State University, 2009
Molecular Beacons Single-stranded nucleic
acids
Stem + Loop
Fluorophore and Quencher
EDANS and DABCYL
Fluorescence resonance energy transfer
Binding changes conformation
Increased distance for molecules= light
Tyagi and Kramer, 1995
MB Real-Time RT-PCR
Kamboj et. al. developed this novel molecular beacon approach
TaqMan Assay Compared in each category
Designed to be:
1. Sensitive
2. Specific
3. Reproducible
Sensitivity• 10-fold serial dilution
I. 7.6 x 10^9 copies
II. 7.6 copies (lower detection limit)
• Standard Curve
SpecificityTested with similar ruminant virus types
I. Bovine Herpes Virus
II. Bovine Immunodeficiency Virus
III. Bovine Foot-and-Mouth Disease
*No detection by the molecular beacons
Reproducibility
• Triplicate of each concentration
• Coefficients of variation (CV) for Cross-Point Cycle (CP)
• CV= st. dev/meanKamboj et. al, 2014
References Garrison, A. R., Alakbarova, S., Kulesh, D. A., Shezmukhamedova,
D., Khodjaev, S., Endy, T. P., & Paragas, J. (2007). Development of a TaqMan®–Minor Groove Binding Protein Assay for the Detection and Quantification of Crimean-Congo Hemorrhagic Fever Virus. The American journal of tropical medicine and hygiene, 77(3), 514-520.
Kamboj, A., Pateriya, A. K., Mishra, A., Ranaware, P., Kulkarni, D. D., & Raut, A. A. (2014). Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India. BioMed research international, 2014.
Tyagi, S., & Kramer, F. R. (1996). Molecular beacons: probes that fluoresce upon hybridization. Nature biotechnology, 14(3), 303-308.