Simultaneous Detection of Marburg Virus and Ebola Virus with TaqMan-based Multiplex Real-time PCR Method

Background: Marburg virus (MARV) and Ebola virus (EBOV) are acute infections with high case fatality rates. It is of great significance for epidemic monitoring and prevention and control of infectious diseases by the development of a rapid, specific, and sensitive quantitative PCR method to detect two pathogens simultaneously.

Methods: Primers and TaqMan probes were designed according to highly conserved sequences of these viruses. Sensitivity, specificity, linear range, limit of detection, and the effects of hemolysis and lipid on real-time qPCR were evaluated.

Results: The linearity of the curve allowed quantification of nucleic acid concentrations in range from 10 to 10  copies/ml per reaction (MARV and EBOV). The limit of detection of EBOV was 40 copies/ml, and MARV was 100 copies/ml. It has no cross-reaction with other pathogens such as hepatitis b virus (HBV), hepatitis c virus (HCV), human papillomavirus (HPV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), cytomegalovirus (CMV), and human immunodeficiency virus (HIV). Repeatability analysis of the two viruses showed that their coefficient of variation (CV) was less than 5.0%. The above results indicated that fluorescence quantitative PCR could detect EBOV and MARV sensitively and specifically.

Conclusions: The TaqMan probe-based multiplex fluorescence quantitative PCR assays could detect EBOV and MARV sensitively specifically and simultaneously.