Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.
Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.
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DOI：
10.1371/journal.pone.0071851
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Stepien CA
中科院分区：
文献类型：
--
作者：
Pierce LR;Willey JC;Palsule VV;Yeo J;Shepherd BS;Crawford EL;Stepien CA
病毒性出血性败血症病毒（VHSV）是世界上最严重的鱼类病原体之一，感染来自欧亚大陆和北美的80多种海洋，淡水和河口鱼类。2003年，五大湖出现了一种新的、毒性特别强的毒株-- IVb，在随后几年的一系列爆发中杀死了许多猎用鱼类，并关闭了州际鱼饵运输。细胞培养是USDA-APHIS批准的诊断方法，需要一个月或更长时间，缺乏敏感性，并且不能量化病毒的数量。因此，我们提出了一种新的，简单的，快速的，高灵敏度的实时定量逆转录PCR（qRT-PCR）检测，采用合成的竞争性模板的内部标准品进行质量控制，以规避假阴性结果。结果表明，高信号-分析物响应（斜率= 1.00±0.02）和线性动态范围跨越7个数量级（R2 = 0.99），范围为6至6，000，000个分子。 发现受感染的鱼类携带的病毒水平范围为1，200，000 VHSV分子/106 actb 1分子，其中1，000是疾病临床症状的粗略截止值。这种新的检测方法快速，廉价，并且比其他已发表的qRT-PCR检测和传统的细胞培养诊断具有更高的准确性。
Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain – IVb – appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R2 = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/106 actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.
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DOI：
10.3758/bf03203630
发表时间：
1996-02-01
期刊：
BEHAVIOR RESEARCH METHODS INSTRUMENTS & COMPUTERS
影响因子：
--
作者：
Erdfelder, E;Faul, F;Buchner, A
通讯作者：
Buchner, A
影响因子：
2.5
作者：
Elsayed, E.;Faisal, M.;Winton, J.
通讯作者：
Winton, J.
影响因子：
1.4
作者：
Hawley, Laura M.;Garver, Kyle A.
通讯作者：
Garver, Kyle A.
影响因子：
3.1
作者：
Chico, V;Gomez, N;Perez, L
通讯作者：
Perez, L
影响因子：
2.5
作者：
Gagne, N.;MacKinnon, A-M;Olivier, G.
通讯作者：
Olivier, G.