Detection of Crimean- Congo Hemorrhagic Fever Using Molecular Beacons Sawyer Ellis PBIO4500 December 2 nd, 2014.

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Presentation transcript:

Detection of Crimean- Congo Hemorrhagic Fever Using Molecular Beacons Sawyer Ellis PBIO4500 December 2 nd, 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

DABCYL and EDANS 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

Sensitivity (Continued) Kamboj et. al, 2014

Sensitivity (Continued) Kamboj et. al, 2014

Specificity Tested 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/mean Kamboj et. al, 2014

Comparison Kamboj 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), 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, Tyagi, S., & Kramer, F. R. (1996). Molecular beacons: probes that fluoresce upon hybridization. Nature biotechnology, 14(3),