Abstract
In order to establish an accurate, ready-to-use assay for simultaneous detection of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV), we developed one duplex TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay, which can be used in human and vector surveillance. First, we selected the primers and FAM-labeled TaqMan-probe specific for WEEV from the consensus sequence of NSP3 and the primers and HEX-labeled TaqMan-probe specific for EEEV from the consensus sequence of E3, respectively. Then we constructed and optimized the duplex real-time RT-PCR assay by adjusting the concentrations of primers and probes. Using a series of dilutions of transcripts containing target genes as template, we showed that the sensitivity of the assay reached 1 copy/reaction for EEEV and WEEV, and the performance was linear within the range of at least 106 transcript copies. Moreover, we evaluated the specificity of the duplex system using other encephalitis virus RNA as template, and found no cross-reactivity. Compared with virus isolation, the gold standard, the duplex real time RT-PCR assay we developed was 10-fold more sensitive for both WEEV and EEEV detection.
Highlights
Eastern equine encephalitis virus (EEEV) and western equine encephalitis virus (WEEV) are both arthropodborne viruses, belonging to the genus Alphavirus, family Togaviridae
Sensitivity and specificity of the duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay The sensitivity of the duplex real-time RT-PCR was conducted as follows: the reaction consisted of 10 μL 2 × reaction buffer, 0.2 μL reverse transcription enzyme, 250 nmol/L primers for WEEV and 500 nmol/L for EEEV, 150 nmol/L probes for WEEV and 250 nmol/L for EEEV
All the samples appeared to be negative and only background fluorescence levels were observed, demonstrating that our real-time RT-PCR detection system was specific for EEEV and WEEV
Summary
Eastern equine encephalitis virus (EEEV) and western equine encephalitis virus (WEEV) are both arthropodborne viruses, belonging to the genus Alphavirus, family Togaviridae. Both viruses are mainly spread in America and transmitted by mosquitoes to equines, birds and humans, causing a febrile disease (including encephalitis) with a significant frequency of fatal outcomes. Compared with traditional methods such as RT-PCR, IFA, ELISA and virus isolation, real-time RT-PCR has the advantage of fast speed and improved sensitivity. It has been developed quickly and become the main method for pathogen detection[3]. We designed the primers and probes of real-time RT-PCR for EEEV and WEEV, and we have established a duplex real time RT-PCR method for simultaneously detection and discrimination of these two viruses
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