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

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

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

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), 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.