Infectious disease - PCR detection of bacteria in seven minutes
Infectious disease - PCR detection of bacteria in seven minutes
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DOI：
10.1126/science.284.5413.449
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发表时间：
1999-04-16
期刊：
影响因子：
56.9
通讯作者：
Milanovich, F
中科院分区：
文献类型：
--
作者：
Belgrader, P;Benett, W;Milanovich, F
识别、监测和隔离传染病爆发的源头有时可能很困难。便携式仪器可在任何可疑地点使用，并能迅速检测和鉴定微生物病原体，这将大大便利这一过程。核酸分析比免疫测定更适合于这种测试，因为可以获得关于特定微生物的毒力，抗生素抗性和流行病学的详细遗传信息。越来越多的这些细菌测试使用聚合酶链反应（PCR），因为它被证明是有效的，灵敏度和可靠性（1，2）。对于现场应用，称为实时PCR的标准PCR的变体是最实用的，因为它不需要耗时的PCR后操作和用平板凝胶和毛细管电泳、与固定化寡核苷酸杂交或质谱法处理反应。实时PCR可以用称为TaqMan的荧光5′-核酸酶测定法（3，4）和分光荧光热循环仪（4）完成。TaqMan是一种使用荧光共振能量转移（FRET）探针的均相PCR检测，该探针通常由5′端的绿色荧光“报告”染料和3′端的橙子“淬灭”染料组成。当探针在PCR期间与扩增子的互补链退火时，Taq聚合酶在引物之一的延伸期间切割探针，并且染料分子被置换和分离。电子激发的报告染料不再受到猝灭剂染料的抑制，并且可以通过荧光检测器监测绿色发射的显着增加。绿色荧光的强度与反应中PCR产物的浓度直接相关。
Identifying, monitoring, and isolating the source of an outbreak of infectious disease can sometimes be difficult. The process would be greatly facilitated by a portable instrument that could be used in any suspect location and was capable of rapidly detecting and characterizing microbial pathogens. Nucleic acid analysis is preferable to immunoassays for such a test because detailed genetic information can be obtained about a particular microbe's virulence, antibiotic resistance, and epidemiology. A growing number of these bacterial tests use polymerase chain reaction (PCR) because of its demonstrated effectiveness, sensitivity, and reliability (1, 2). For field use, a variation of standard PCR called real-time PCR is the most practical, because it does not require time-consuming post-PCR manipulation and processing of the reaction with slab gel and capillary electrophoresis, hybridization to immobilized oligonucleotides, or mass spectrometry.Real-time PCR can be accomplished with the fluorogenic 5′-nuclease assay called TaqMan (3, 4) and a spectrofluorometric thermal cycler (4). TaqMan is a homogenous PCR test that uses a fluorescence resonance energy transfer (FRET) probe typically consisting of a green fluorescent “reporter” dye at the 5′-end and an orange “quencher” dye at the 3′-end. When the probe anneals to a complementary strand of an amplicon during PCR, Taq polymerase cleaves the probe during extension of one of the primers, and the dye molecules are displaced and separated. The electronically excited reporter dye is no longer suppressed by the quencher dye, and the significant increase in green emission can be monitored by a fluorescence detector. The intensity of the green fluorescence directly correlates with the concentration of PCR product in the reaction.