Highly sensitive and multiplex detection of nine potential bioterrorism viral agents in a single reaction by multiplex probe amplification (MPA) with melting curve analysis.

UNLABELLED: The rapid and accurate detection of highly pathogenic viruses is critical for public health, especially in the context of potential bioterrorism threats. This study presents a novel multiplex probe amplification (MPA) assay combined with melting curve analysis for the simultaneous detection of nine high-threat viral agents: Ebola virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, Chikungunya virus, Monkeypox virus, Eastern equine encephalitis virus, Tick-borne encephalitis virus, and Venezuelan equine encephalitis virus. The MPA-nine-viruses assay utilizes three fluorescence channels and unique melting temperatures to differentiate between targets, achieving high sensitivity with a limit of detection as low as 1.5-15 copies/μL. The MPA-nine-viruses was highly specific and distinguished mixed targets accurately, even for the nine viruses' mixture. The assay was validated using 392 simulated samples (including serum, swabs, mosquito vectors, and soil samples) and 22 clinical samples, demonstrating 100% accuracy. The MPA-nine-viruses assay offers a cost-effective, high-throughput solution for large-scale screening and is poised to play a crucial role in the prevention and control of outbreaks and bioterrorism events involving these pathogens.

IMPORTANCE: Ebola virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, Chikungunya virus, Monkeypox virus, Eastern equine encephalitis virus, Tick-borne encephalitis virus, and Venezuelan equine encephalitis virus can cause severe infections and diseases, such as hemorrhagic fever, encephalitis, meningitis, and chikungunya fever. These viruses pose significant threats to public health due to their high infectivity and mortality rates, and they could potentially be used as bioterrorism agents. Early detection is the most critical step for effective prevention and control of infection. However, there is no sensitive nucleic acid detection method for the nine high-threat viral agents tested simultaneously. In this study, we developed multiplex probe amplification (MPA) with Melting Curve PCR method, named MPA-nine-viruses, which overcame the limitation of the available fluorescence channel number and realized simultaneous detection of nine high-threat viral agents in a single reaction, with high sensitivity and specificity for the targets.