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学科主题: Biochemical Research Methods; Biotechnology & Applied Microbiology; Virology
题名: An improved RT-PCR assay for rapid and sensitive detection of grass carp reovirus
作者: Zhang, Lanlan1; Luo, Qing2; Fang, Qin1; Wang, Yaping2
通讯作者: Fang, Q, Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China
关键词: dsRNA virus ; Aquareovirus ; Grass carp reovirus ; RNA extraction ; RT-PCR rapid detection
刊名: JOURNAL OF VIROLOGICAL METHODS
发表日期: 2010-10-01
DOI: 10.1016/j.jviromet.2010.06.009
卷: 169, 期:1, 页:28-33
收录类别: SCI
文章类型: Article
部门归属: [Zhang, Lanlan; Fang, Qin] Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China; [Luo, Qing; Wang, Yaping] Chinese Acad Sci, Inst Hydrobiol, Wuhan 430072, Peoples R China
WOS标题词: Science & Technology ; Life Sciences & Biomedicine
资助者: National Basic Research Program of China [2009CB118701]; National Natural Scientific Foundation of China [30871940, 30671615]
类目[WOS]: Biochemical Research Methods ; Biotechnology & Applied Microbiology ; Virology
研究领域[WOS]: Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Virology
摘要: An improved simple, rapid and sensitive method for detecting grass carp reovirus (GCRV) based on RT-PCR was developed by combining an advanced RNA extraction technique and targeting segment 10 as a template. The results indicate that highly efficient RT-PCR amplification of GCRV genome segments can be obtained using column-extracted RNA as a template, which is suitable not only for full-length gene amplification up to a size of 1.5 kb, but also for partial genome detection. Moreover, by targeting the highly divergent segment 10, the sensitivity of RT-PCR detection is improved significantly; as little as 1.0 fg of the 515 bp S10 dsRNA can be detected by one-step RT-PCR amplification. Furthermore, this method exhibits good reproducibility and specificity, and no amplicons were observed when RNA fragments other than those from GCRV were used as templates. The entire detection process can be completed within 4-5 h from RNA extraction, much faster than methods reported previously. Overall, the improved detection technique may be applied for rapid diagnosis of GCRV or other dsRNA viruses. (c) 2010 Elsevier B.V. All rights reserved.
英文摘要: An improved simple, rapid and sensitive method for detecting grass carp reovirus (GCRV) based on RT-PCR was developed by combining an advanced RNA extraction technique and targeting segment 10 as a template. The results indicate that highly efficient RT-PCR amplification of GCRV genome segments can be obtained using column-extracted RNA as a template, which is suitable not only for full-length gene amplification up to a size of 1.5 kb, but also for partial genome detection. Moreover, by targeting the highly divergent segment 10, the sensitivity of RT-PCR detection is improved significantly; as little as 1.0 fg of the 515 bp S10 dsRNA can be detected by one-step RT-PCR amplification. Furthermore, this method exhibits good reproducibility and specificity, and no amplicons were observed when RNA fragments other than those from GCRV were used as templates. The entire detection process can be completed within 4-5 h from RNA extraction, much faster than methods reported previously. Overall, the improved detection technique may be applied for rapid diagnosis of GCRV or other dsRNA viruses. (c) 2010 Elsevier B.V. All rights reserved.
关键词[WOS]: POLYMERASE-CHAIN-REACTION ; GENUS AQUAREOVIRUS ; FAMILY REOVIRIDAE ; DIAGNOSTIC ASSAY ; FISH ; VIRUS ; HYBRIDIZATION ; IDENTIFICATION ; GENOGROUP ; RNA
语种: 英语
WOS记录号: WOS:000282398600005
ISSN号: 0166-0934
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/342005/13579
Appears in Collections:鱼类生物学及渔业生物技术研究中心_期刊论文

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作者单位: 1.Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China
2.Chinese Acad Sci, Inst Hydrobiol, Wuhan 430072, Peoples R China

Recommended Citation:
Zhang, Lanlan; Luo, Qing; Fang, Qin; Wang, Yaping.An improved RT-PCR assay for rapid and sensitive detection of grass carp reovirus,JOURNAL OF VIROLOGICAL METHODS,2010,169(1):28-33
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