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Related Experiment Videos

Real-time PCR assay to detect smallpox virus.

M Sofi Ibrahim1, David A Kulesh, Sharron S Saleh

  • 1The United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702, USA. Sofi.Ibrahim@det.amedd.army.mil

Journal of Clinical Microbiology
|August 9, 2003
PubMed
Summary

A new assay rapidly detects smallpox virus DNA with high sensitivity and specificity. This TaqMan-based test offers early detection capabilities for orthopoxvirus infections, crucial for public health preparedness.

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Area of Science:

  • Molecular Biology
  • Virology
  • Infectious Diseases

Background:

  • Smallpox virus poses a significant public health threat, necessitating rapid and accurate diagnostic tools.
  • Existing diagnostic methods may lack the sensitivity or speed required for timely detection and response.
  • The orthopodox hemagglutinin gene is a conserved target for poxvirus detection.

Purpose of the Study:

  • To develop and validate a highly sensitive and specific assay for the rapid detection of smallpox virus DNA.
  • To evaluate the assay's performance on two common real-time PCR platforms: Smart Cycler and LightCycler.
  • To assess the assay's utility for early detection of variola virus infections.

Main Methods:

  • Development of a TaqMan-based real-time PCR assay targeting the orthopoxvirus hemagglutinin gene.

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  • Testing the assay with purified viral DNA and DNA from infected cell cultures and tissues.
  • Evaluation in a blinded study using 322 coded samples, including various orthopoxviruses and other agents.

    • Main Results:

    • The assay demonstrated a limit of detection of approximately 25 copies of smallpox virus DNA.
    • Specificities were 98.3% on the Smart Cycler and 95.7% on the LightCycler.
    • Clinical sensitivities were 96.1% (Smart Cycler) and 99.5% (LightCycler), with 100% detection of purified variola virus DNA.

    Conclusions:

    • The developed assay is highly sensitive and specific for smallpox virus DNA detection.
    • The assay performs effectively on both Smart Cycler and LightCycler platforms.
    • This diagnostic tool holds potential for early detection of smallpox virus, aiding in bioterrorism preparedness and response.