Background
Ectromelia virus (ECTV) is an important pathogen that could cause the acute lethal viral disease known as mousepox. This disease has severe epidemic feature with high mortality and significant harm. In order to prevent and control the occurrence of mousepox, it is essential to develop a fast, sensitive diagnostic tool for rapid and specific detection of infectious Ectromelia virus. The aim of this study was to develop a TaqMan-minor groove binder (TaqMan -MGB) probe-based fluorescence quantitative real-time polymerase chain reaction (qPCR) assay for rapid detection of Ectromelia virus.
Methods
Plasmid containing the sequence of HA gene (168155-168239 nt) was constructed as ECTV-DNA standard for quantitative analysis. In this study, a set of reliable and specific qPCR primers and a TaqMan MGB probe were used to detect Ectromelia virus. The interested sequence contained in the plasmid and in clinical specimens was quantitatively measured.
Results
The TaqMan-MGB probe qPCR assay was shown to be 100% specific to test a panel of seventeen organisms consisting of ECTV, other orthopoxvirus species, non-orthopoxvirus species, bacteria, fungi, parasites and cells. The technology was demonstrated to be highly sensitive, allowing a precise ECTV DNA quantitation over a range of ten orders of magnitude (from 100 to 1010 copies of standard DNA), and the limit of detection (LOD) of the assay was determined to be 3 copies of target DNA. The reproducibility of standard curve was excellent as the variation coefficient (%CV) for each point was less than 3%. Measurements of linearity such as R2, S, y, and Eff(%) did not vary significantly among the test (%CV<3%) suggesting high accuracy. The TaqMan MGB probe qPCR assay was successfully applied to quantifiable detection of viral genomic load in clinical specimens, and confirmed by using ELISA, conventional PCR and sequence analysis. The assay described in this report generates complete result in 2 h and can be used as a rapid diagnostic tool.
Conclusion
Our study demonstrated that this new TaqMan-MGB probe qPCR assay is an accurate, rapid and reliable method that can be used for the quantitative detection of ECTV, especially allowing the detection of low loading of ECTV in clinical specimens. It also could be applied to quantifiable detection of viral loading in animal origin products and biological products, food and drug safety inspection, environmental monitoring and epidemiology investigation. It is the first report to describe a TaqMan-MGB probe qPCR for specific diagnosis of mousepox infection.
Author(s): Zheng-qin Gao, and Bing-fei Yue