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首页> 美国卫生研究院文献>Journal of Clinical Microbiology >Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms
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Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms

机译：在Roche LightCycler和Cepheid Smart Cycler平台上通过实时3′-小沟结合剂TaqMan检测天花和泛正痘病毒

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We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3′-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 × 107, 1.24 × 105, 1.24 × 103, and 1.24 × 101 genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However, all five assays had nearly 100% sensitivity on both machines with samples above the LOD (>12 gene copies). These real-time PCR assays represent a battery of tests to screen for and confirm the presence of variola virus DNA. The early detection of a smallpox outbreak is crucial whether the incident is an act of bioterrorism or an accidental occurrence.
机译：我们设计，优化和广泛测试了几种灵敏且实时的实时PCR检测方法，用于快速检测天花和全正痘病毒DNA。该测定基于TaqMan 3'-小沟结合剂化学性质，在快速循环的Roche LightCycler和Cepheid Smart Cycler平台上进行。将血凝素（HA）J7R，B9R和B10R基因用作天花病毒特异性检测的靶标，并将HA和DNA聚合酶E9L基因用作全正痘病毒检测的靶标。在美国陆军传染病研究所（USAMRIID），针对一组正痘病毒DNA（基因组和克隆）测试了五种正痘病毒检测方法。结果表明，每种测定法都能够检测合适的克隆基因和基因组DNA。该测定显示在USAMRIID细菌交叉反应性面板中与78个DNA没有交叉反应性。每种测定的检出限（LOD）确定为目标DNA的12到25个拷贝之间。在LightCycler和Smart Cycler上的疾病控制和预防中心（CDC）上，还对DNA盲板进行了测定。该小组由八种不同的天花病毒分离株，五种非天花病毒正痘病毒分离株，两种水痘带状疱疹病毒分离株和一种单纯疱疹病毒分离株组成。每个样品分别以2.5 ng，25 pg，250 fg和2.5 fg一式三份进行测试，分别代表1.24×10 7 ，1.24×10 5 ，1.24×10 < sup> 3 和1.24×10 1 基因组当量。结果表明，当针对USAMRIID面板和CDC盲面板进行测试时，五种测定法中的每一种都是100％特异性的（无假阳性）。使用CDC盲板，LightCycler能够检测96.2％的正痘病毒DNA和93.8％的天花病毒DNA。 Smart Cycler能够检测92.3％的正痘病毒DNA和75-93.8％的天花病毒DNA。但是，所有两种测定在两台机器上的样品均高于LOD（> 12个基因拷贝）时，其灵敏度均接近100％。这些实时PCR分析代表了一系列测试，以筛选并确认天花病毒DNA的存在。无论事件是生物恐怖主义行为还是偶然事件，及早发现天花爆发都是至关重要的。

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