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A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral
Sanchita Das1, Mark S Rundell2, Aashiq H Mirza2
1Department of Medicine, Division of Infectious Diseases, Weill Medical College of Cornell University, New York, New York, United States of America.
Abstract:
CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).
Insights
A new multiplex assay rapidly detects Category A infectious agents like viral hemorrhagic fever (VHF) viruses and variola virus (smallpox). This rapid detection is crucial for public health preparedness and preventing outbreaks.
Area of Science:
- Virology
- Public Health
- Molecular Diagnostics
Background:
- Category A infectious agents, including viral hemorrhagic fever (VHF) viruses and variola virus, pose significant national security and public health risks.
- Early and accurate detection is critical for outbreak containment, control measures, and mitigating high morbidity and mortality.
Purpose of the Study:
- To develop and evaluate a multiplex detection assay for the simultaneous identification of Category A infectious agents.
- To distinguish these high-risk pathogens from other emerging zoonoses and flaviviruses.
Main Methods:
- Development of a multiplex assay utilizing species-specific PCR primers and ligase detection reaction (LDR).
- Detection of fluorescently labeled ligation products on a universal array for simultaneous pathogen identification.
Main Results:
- The assay successfully detected all 32 tested VHF virus isolates and variola virus strains.
- The assay demonstrated no cross-reactivity with other zoonotic viruses like monkeypox or cowpox, or tested flaviviruses.
Conclusions:
- The developed multiplex LDR assay provides a sensitive and specific tool for the simultaneous detection of critical Category A pathogens.
- This assay is a valuable advancement for public health preparedness and rapid response to potential bioterrorism events.
