TaqMan Multiplex Real-time PCR Assay for Crimean-Congo Hemorrhagic Fever Virus Diagnosis Using Armored RNA Technology

Background: Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly lethal that causes in humans and endemic many countries, including Iran. Therefore, fast, accurate, reliable diagnosis crucial for patient management outbreak control. Objectives: This study aims to optimize TaqMan multiplex real-time RT-PCR the rapid specific of CCHFV. Methods: In this study, L (NC_005301.3) S (NC_005302.1) fragments were used as reference sequences blast analysis. The sequence segments CCHFV with more than 90% identity from different areas downloaded Genbank database. Primers probes designed based on best-conserved regions segments. To construct plasmid, 1751 bp fragment MS2 phage was previously amplified using cloning primers inserted into pET-32a plasmid. CCHFV, which 110 135 bp, respectively, downstream HindIII NotI. Viral-like particles (VLPs) produced Escherichia coli, strain BL-21(DE3), presence 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). stability VLP confirmed ribonuclease enzyme. fabrication VLPs approved by transmission electron microscopy (TEM) negative staining (1% phosphotungstic acid). validate specificity sequences, we compared them NCBI database tested experimentally extracted DNA RNA samples healthy subjects an infectious panel. Results: showed complete resistance enzyme, TEM results correctly produced. completely limit detection (LOD) assay genes one copy per µL. Conclusions: amplifying due its design conserved have minimal variability high specificity.