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利用光纤表面等离子体共振PCR技术鉴定和定量芹菜过敏原

Identification and Quantification of Celery Allergens Using Fiber Optic Surface Plasmon Resonance PCR.

作者信息

Daems Devin, Peeters Bernd, Delport Filip, Remans Tony, Lammertyn Jeroen, Spasic Dragana

机构信息

KU Leuven-University of Leuven, BIOSYST-MeBioS, Willem de Croylaan 42, B-3001 Leuven, Belgium.

FOx Diagnostics NV-Veldstraat 120, B-9140 Temse, Belgium.

出版信息

Sensors (Basel). 2017 Jul 31;17(8):1754. doi: 10.3390/s17081754.

Abstract

: Accurate identification and quantification of allergens is key in healthcare, biotechnology and food quality and safety. Celery () is one of the most important elicitors of food allergic reactions in Europe. Currently, the golden standards to identify, quantify and discriminate celery in a biological sample are immunoassays and two-step molecular detection assays in which quantitative PCR (qPCR) is followed by a high-resolution melting analysis (HRM). In order to provide a DNA-based, rapid and simple detection method suitable for one-step quantification, a fiber optic PCR melting assay (FO-PCR-MA) was developed to determine different concentrations of celery DNA (1 pM-0.1 fM). The presented method is based on the hybridization and melting of DNA-coated gold nanoparticles to the FO sensor surface in the presence of the target gene (mannitol dehydrogenase, ). The concept was not only able to reveal the presence of celery DNA, but also allowed for the cycle-to-cycle quantification of the target sequence through melting analysis. Furthermore, the developed bioassay was benchmarked against qPCR followed by HRM, showing excellent agreement (² = 0.96). In conclusion, this innovative and sensitive diagnostic test could further improve food quality control and thus have a large impact on allergen induced healthcare problems.

摘要

过敏原的准确鉴定和定量在医疗保健、生物技术以及食品质量与安全领域至关重要。芹菜是欧洲引发食物过敏反应的最重要诱因之一。目前,在生物样本中鉴定、定量和区分芹菜的金标准是免疫测定法和两步分子检测法,其中定量聚合酶链反应(qPCR)之后是高分辨率熔解分析(HRM)。为了提供一种基于DNA的、快速且简单的适用于一步定量的检测方法,开发了一种光纤PCR熔解分析(FO-PCR-MA)来测定不同浓度的芹菜DNA(1 pM - 0.1 fM)。所提出的方法基于在靶基因(甘露醇脱氢酶)存在的情况下,DNA包被的金纳米颗粒与光纤传感器表面的杂交和熔解。该概念不仅能够揭示芹菜DNA的存在,还能通过熔解分析对靶序列进行逐循环定量。此外,将开发的生物测定法与qPCR之后的HRM进行了基准测试,显示出极佳的一致性(R² = 0.96)。总之,这种创新且灵敏的诊断测试可进一步改善食品质量控制,从而对过敏原引发的医疗问题产生重大影响。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/bfaa/5579924/7062bc2b51d2/sensors-17-01754-g001.jpg

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