Rapid typing of influenza viruses using super high-speed quantitative real-time PCR.
Rapid typing of influenza viruses using super high-speed quantitative real-time PCR.
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DOI：
10.1016/j.jviromet.2011.08.015
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发表时间：
2011-12
影响因子：
3.1
通讯作者：
Shibasaki F
中科院分区：
文献类型：
--
作者：
Sakurai A;Nomura N;Nanba R;Sinkai T;Iwaki T;Obayashi T;Hashimoto K;Hasegawa M;Sakoda Y;Naito A;Morizane Y;Hosaka M;Tsuboi K;Kida H;Kai A;Shibasaki F
建立了►A超高速定量聚合酶链式反应(SHRT-PCR法)。RT反应和聚合酶链式反应(40个循环)可在20分钟内完成。►尽管反应速度快，但其灵敏度和特异度与传统的定量逆转录聚合酶链式反应体系相当。►可用于临床标本的检测。开发快速和灵敏的流感病毒检测系统是控制未来流行和大流行的高度优先事项。定量实时聚合酶链式反应经常用于检测各种病毒；然而，它需要每次运行2小时以上。检测分析采用根据新设计的加热系统开发的超高速RT-PCR(SHRT-PCR)。新方法使用高速反应(18个S/周期；40个周期不到20个周期)来对流感病毒进行分型。该方法对培养上清液中病毒的检测下限为1个拷贝/反应和10个−-1空斑形成单位/反应。用SHRT-PCR方法对东京都公共卫生研究所分离的86株流感病毒进行检测，结果显示对甲型、乙型流感病毒和猪源流感病毒的敏感性和特异性均为100%。从门诊患者咽粘膜采集的27份拭子也进行了检测，免疫层析检测显示流感病毒呈阳性迹象；SHRT-PCR和免疫层析的结果显示阳性和阴性结果均为100%一致。SHRT-PCR的快速反应时间和高灵敏度使该技术非常适合于监测流行病和大流行前的流感暴发。
► A super high speed qRT-PCR (SHRT-PCR) was developed. RT reaction and PCR (40 cycles) can be completed in less than 20 min. ► Despite the high speed, the sensitivity and specificity of the SHRT-PCR are comparable to conventional qRT-PCR system. ► The SHRT-PCR can be used to test clinical samples. The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2 h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18 s/cycle; 40 cycles in less than 20 min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10−1 plaque-forming unit/reaction for viruses in culture supernatants during 20 min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks.
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影响因子：
8.8
作者：
Chan, K. H.;Lam, S. Y.;Peiris, J. S. M.
通讯作者：
Peiris, J. S. M.
影响因子：
3.1
作者：
Oleksiewicz, MB;Donaldson, AI;Alexandersen, S
通讯作者：
Alexandersen, S
影响因子：
3.1
作者：
Miyagawa, Eiji;Kogaki, Hiroyuki;Kida, Hiroshi
通讯作者：
Kida, Hiroshi
影响因子：
9.4
作者：
Bose, Michael E.;Beck, Eric T.;Henrickson, Kelly J.
通讯作者：
Henrickson, Kelly J.
影响因子：
1.2
作者：
Bai, GR;Sakoda, Y;Kida, H
通讯作者：
Kida, H