Multiplex nucleic acid polymerase assay for eight severe hemorrhagic fever viruses based on dual-probe hybridization and melting curve analysis.

Abstract

Background: In recent years, frequent outbreaks of infectious diseases caused by hemorrhagic fever viruses have posed serious threats to global public health. The pathogens are variable and highly infectious, such as Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Marburg marburgvirus (MARV), Lassa mammarenavirus (LASV), Rift Valley fever phlebovirus (RVFV), Sin Nombre orthohantavirus (SNV), etc. To improve the efficiency of pathogen detection, a method for simultaneous screening multiplex targets is in a great demand.

Methods: Utilizing dual-probe hybridization and melting curve analysis, a multiplex nucleic acid polymerase assay for eight hemorrhagic fever viruses test (named the MPA-eight-virus assay) was developed in this study. The sensitivity for each target was improved by optimizing primer and probe selection as well as amplification conditions; the usability of MPA-eight-virus assay was validated by simulated samples preparation and test.

Results: The MPA-eight-virus assay achieved high sensitivity and specificity for the targets, with a limit of detection (LOD) of 1.83-691.00 copies/µL for all eight targets; Notably, the LOD for MARV was 1.83 copies/µL and that for SNV was 9.32 copies/µL.

Conclusions: The MPA-eight-virus assay is high throughput, time-saving, accurate, and cost-effective, making it potentially useful for prevention and control of severe viral hemorrhagic fever.