Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents
Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents
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DOI：
10.1016/j.jviromet.2008.07.018
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发表时间：
2008-11-01
影响因子：
3.1
通讯作者：
Ibrahim, Sofi
中科院分区：
文献类型：
--
作者：
Aitichou, Mohamed;Saleh, Sharron;Ibrahim, Sofi
建立了一种基于干燥聚合酶链式反应试剂的实时、多重聚合酶链式反应分析方法。用FAM(6-羧基荧光素)标记的探针在一次PCR反应中只能特异性地检测到天花病毒，而用TET(6-羧基四甲基罗丹明)标记的探针可以检测到山痘、牛痘、猴痘和牛痘病毒。可检测到约25个拷贝的克隆天花病毒DNA和50个拷贝的正痘病毒基因组DNA，重复性较好。该方法的动态范围为7个数量级，相关系数大于0.97。对100份含有多种细菌和病毒核酸的样品进行检测，其灵敏度和特异度分别为96%和98%。该方法的检出限、灵敏度和特异度与标准的实时荧光聚合酶链式反应湿试剂法相当。在本试验中采用多重形式允许同时区分天花病毒和其他密切相关的正痘病毒。此外，在实时聚合酶链式反应分析中使用干燥试剂是简化这类分析并允许在冷藏不易获得的地区使用的重要一步。(C)2008爱思唯尔B.V.保留所有权利。
A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible. (C) 2008 Elsevier B.V. All rights reserved.