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A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses

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 hum...

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Autores principales: Kang, Xiaoping, Li, Yuchang, Liu, Hong, Lin, Fang, Cai, Xuyu, Sun, Tingting, Chang, Guohui, Zhu, Qingyu, Yang, Yinhui
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2974676/
https://www.ncbi.nlm.nih.gov/pubmed/20977706
http://dx.doi.org/10.1186/1743-422X-7-284
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author Kang, Xiaoping
Li, Yuchang
Liu, Hong
Lin, Fang
Cai, Xuyu
Sun, Tingting
Chang, Guohui
Zhu, Qingyu
Yang, Yinhui
author_facet Kang, Xiaoping
Li, Yuchang
Liu, Hong
Lin, Fang
Cai, Xuyu
Sun, Tingting
Chang, Guohui
Zhu, Qingyu
Yang, Yinhui
author_sort Kang, Xiaoping
collection PubMed
description 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 10(6 )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.
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spelling pubmed-29746762010-11-06 A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses Kang, Xiaoping Li, Yuchang Liu, Hong Lin, Fang Cai, Xuyu Sun, Tingting Chang, Guohui Zhu, Qingyu Yang, Yinhui Virol J Short Report 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 10(6 )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. BioMed Central 2010-10-26 /pmc/articles/PMC2974676/ /pubmed/20977706 http://dx.doi.org/10.1186/1743-422X-7-284 Text en Copyright ©2010 Kang et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Short Report
Kang, Xiaoping
Li, Yuchang
Liu, Hong
Lin, Fang
Cai, Xuyu
Sun, Tingting
Chang, Guohui
Zhu, Qingyu
Yang, Yinhui
A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses
title A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses
title_full A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses
title_fullStr A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses
title_full_unstemmed A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses
title_short A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses
title_sort duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses
topic Short Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2974676/
https://www.ncbi.nlm.nih.gov/pubmed/20977706
http://dx.doi.org/10.1186/1743-422X-7-284
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