Detection of pathogen-specific antibodies by loop-mediated isothermal amplification.
Detection of pathogen-specific antibodies by loop-mediated isothermal amplification.
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通过环介导的等温扩增检测病原体特异性抗体。
DOI：
10.1128/cvi.00811-14
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
Grab,DennisJ
中科院分区：
文献类型：
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作者：
Burbulis,IanE;Yamaguchi,Kumiko;Nikolskaia,OlgaV;Prigge,SeanT;Magez,Stefan;Bisser,Sylvie;Reller,MeganE;Grab,DennisJ
环介导等温扩增 (LAMP) 是一种酶促复制 DNA 的方法，由于其高灵敏度、特异性、速度和技术要求（等温条件），对于护理点 (POC) 的临床诊断具有很大的实用性。在这里，我们通过创建将寡核苷酸（LAMP 模板）连接到 IgG 抗体的蛋白质-DNA 融合体（此处称为“LAMPole”），采用 LAMP 来测量蛋白质分析物。该融合体由与 IgG 结合多肽（蛋白质 L/G 结构域）共价结合的 DNA 元件组成。在我们的平台中，LAMP 有望为 POC 临床诊断中的 LAMPole 扩增提供最合适的方法，而定量 PCR 更适合基于实验室的抗原特异性 IgG 丰度定量。作为概念证明，我们通过实时量化溶液浊度的变化来测量对原生动物寄生虫的血清学反应。我们观察到，接种疫苗的小鼠血清与对照小鼠的血清以及临床患者样本与对照小鼠的血清信号存在 >6 个对数倍的差异。我们断言，LAMPoles 将有助于提高测量蛋白质的灵敏度，无论是在临床实验室还是在现场环境中，从而改善各种感染的急性诊断。
Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a “LAMPole”) that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections.