Detection of biological threat agents by real-time PCR: Comparison of assay performance on the RAPID, the LightCycler, and the smart cycler platforms
Detection of biological threat agents by real-time PCR: Comparison of assay performance on the RAPID, the LightCycler, and the smart cycler platforms
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DOI：
10.1373/clinchem.2005.052522
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发表时间：
2006-01-01
影响因子：
9.3
通讯作者：
Norwood, DA
中科院分区：
文献类型：
--
作者：
Christensen, DR;Hartman, LJ;Norwood, DA
背景：生物威胁因子的快速检测对于及时治疗至关重要。荧光PCR提供了一种快速，灵敏，特异的工具，这些代理商的分子鉴定。我们比较了爱达荷州技术公司的7种生物威胁剂的测定性能。R.A.P.I. D (R)方法：设计引物和双标记荧光探针，分别检测炭疽杆菌、布鲁氏菌、肉毒梭菌、贝氏柯克斯体、土拉弗朗西斯菌、金黄色葡萄球菌和鼠疫耶尔森菌。通过使用爱达荷州技术缓冲液和补充有Invitrogen Platinum（R）Taq DNA聚合酶的脱氧核苷酸三磷酸来优化DNA扩增测定，并且随后在R.A.P.I.D.上测试灵敏度和特异性，结果：检测限实验表明，在测试的平台之间，测定性能是相当的。用含有60个DNA的一般细菌核酸交叉反应性板和含有感兴趣的生物体的最近邻的试剂特异性板进行的排他性和包容性测试表明，所有检测试剂对其预期靶标均具有特异性。通过少量补充，例如向爱达荷州技术缓冲液中添加Smart Cycler添加剂试剂，对来自生物威胁物的DNA模板的测定在所有3个平台上显示出相似的性能、灵敏度和特异性。(c)2006年美国临床化学协会。
Background: Rapid detection of biological threat agents is critical for timely therapeutic administration. Fluorogenic PCR provides a rapid, sensitive, and specific tool for molecular identification of these agents. We compared the performance of assays for 7 biological threat agents on the Idaho Technology, Inc. R.A.P.I.D.(R), the Roche LightCycler (R), and the Cepheid Smart Cycle (R).Methods: Real-time PCR primers and dual-labeled fluorogenic probes were designed to detect Bacillus anthracis, Brucella species, Clostridium botulinum, Coxiella burnetii, Francisella tularensis, Staphylococcus aureus, and Yersinia pestis. DNA amplification assays were optimized by use of Idaho Technology buffers and deoxynucleotide triphosphates supplemented with Invitrogen Platinum (R) Taq DNA polymerase, and were subsequently tested for sensitivity and specificity on the R.A.P.I.D., the LightCycler, and the Smart Cycler.Results: Limit of detection experiments indicated that assay performance was comparable among the platforms tested. Exclusivity and inclusivity testing with a general bacterial nucleic acid cross-reactivity panel containing 60 DNAs and agent-specific panels containing nearest neighbors for the organisms of interest indicated that all assays were specific for their intended targets.Conclusion: With minor supplementation, such as the addition of Smart Cycler Additive Reagent to the Idaho Technology buffers, assays for DNA templates from biological threat agents demonstrated similar performance, sensitivity, and specificity on all 3 platforms. (c) 2006 American Association for Clinical Chemistry.