Home › Microbiology › Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis
Microbiology JoVE (Open Access) Citable · DOI

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

DOI: 10.3791/59709-v
What you'll learn
  • ✓ Prepare RNA dilution series for real-time RT-PCR calibration standards
  • ✓ Execute real-time PCR master mix setup and thermal cycling protocol
  • ✓ Interpret fluorescence data and distinguish positive lyssavirus detection
  • ✓ Diagnose rabies from ante-mortem and post-mortem clinical samples
Protocol

Biopharma Insights This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.

Difficulty
intermediate
Total time
~3-4 hours per sample batch (RNA extraction to result interpretation)
Biosafety
BSL-2

Steps

1
Prepare RNA dilution series for standards

Create serial dilutions of extracted rabies RNA to establish a standard curve for quantification and validation of real-time PCR sensitivity.

▶ 00:58
2
Set up and run real-time PCR reaction

Prepare master mix with dsDNA intercalating dye, distribute to wells, add template RNA, and execute thermal cycling protocol on real-time PCR instrument.

▶ 01:22
3
Analyze fluorescence curves and Ct values

Review amplification plots, determine threshold cycles, and apply data analysis software to identify positive and negative lyssavirus samples.

▶ 03:33
4
Interpret pan-lyssavirus RT-PCR results

Evaluate final amplification data to confirm rabies virus detection from clinical samples and distinguish true positives from negative controls.

▶ 04:28
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