生物传感器技术和表面等离子体共振用于实时检测聚合酶链反应扩增的HIV-1基因组序列

Nicoletta Bianchi , Cristina Rutigliano , Marina Tomassetti , Giordana Feriotto , Francesco Zorzato , Roberto Gambari
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引用次数: 63

摘要

背景:用于生物特异性相互作用分析的生物传感器技术的最新发展使得通过表面等离子体共振(SPR)实时监测各种分子反应成为可能。如果配体是生物素化的单链DNA,该技术可以监测DNA-DNA杂交。这种方法可能对病毒学有很大的兴趣,因为杂交步骤通常需要确认分子诊断的特异性。目的:探讨利用生物传感器和SPR技术对人类免疫缺陷病毒I型(HIV-1)进行实时分子诊断的可行性。研究设计:用BIAcore生物传感器检测固定在传感器芯片上的生物素化HIV-1寡核苷酸探针与非对称聚合酶链反应(PCR)获得的单链DNA的特异性杂交。结果:将不对称PCR直接注射到携带内部HIV-1寡核苷酸探针的传感器芯片上,可以使用生物传感器技术通过SPR检测杂交。这使我们能够应用实时、一步、无放射性的方案来证明通过PCR扩增HIV-1基因组序列的特异性。结论:本研究描述的HIV-1检测程序简单、快速(PCR和SPR分析只需30分钟)、可重复性好,可作为基于实验室工作站和生物传感器的DNA分离、PCR反应制备和PCR产物分析自动化诊断系统的组成部分。
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Biosensor technology and surface plasmon resonance for real-time detection of HIV-1 genomic sequences amplified by polymerase chain reaction

Background: The recent development of biosensor technologies for biospecific interaction analysis enables the monitoring of a variety of molecular reactions in real time by surface plasmon resonance (SPR). If the ligand is a biotinylated single stranded DNA, this technology could monitor DNA-DNA hybridization. This approach could be of great interest in virology, since the hybridization step is oftenly required to confirm specificity of molecular diagnosis.

Objectives: To determine whether real-time molecular diagnosis of human immunodeficiency virus type I (HIV-1) could be performed using biosensors and SPR technology.

Study design: Specific hybridization of a biotinylated HIV-1 oligonucleotide probe immobilized on a sensor chip to single stranded DNA obtained by asymmetric polymerase-chain reaction (PCR) was determined using the BIAcore biosensor.

Results: Direct injection of asymmetric PCR to a sensor chip carrying an internal HIV-1 oligonucleotide probe allows detection of hybridization by SPR using biosensor technology. This enabled us to apply a real-time, one-step, non-radioactive protocol to demonstrate the specificity of amplification of HIV-1 genomic sequences by PCR.

Conclusion: The procedure described in this study for HIV-1 detection is simple, fast (PCR and SPR analyses take 30 min), reproducible and could be proposed as an integral part of automated diagnostic systems based on the use of laboratory workstations and biosensors for DNA isolation, preparation of PCR reactions and analysis of PCR products.

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