tailieunhanh - Báo cáo y học: " A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses?"

Tuyển tập báo cáo các nghiên cứu khoa học quốc tế ngành y học dành cho các bạn tham khảo đề tài: A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses | Kang et al. Virology Journal 2010 7 284 http content 7 1 284 VIROLOGY JOURNAL SHORT REPORT Open Access A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses Xiaoping Kang Yuchang Li Hong Liu Fang Lin Xuyu Cai Tingting Sun Guohui Chang Qingyu Zhu Yinhui Yang 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. Introduction Eastern equine encephalitis virus EEEV and western equine encephalitis virus WEEV are both arthropod-borne 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. The fatality rate of EEEV is .