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

ABSTRACT: Abstract: Accurate identification and quantification of allergens is key in healthcare, biotechnology and food quality and safety. Celery (Apium graveolens) 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, Mtd). 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 (R² = 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.

SUBMITTER: Daems D

PROVIDER: S-EPMC5579924 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThrombotic thrombocytopenic purpura (TTP) is characterized by severe ADAMTS-13 activity deficiency (<10%). Diagnostic testing is challenging because of unavailability, high cost, and expert technician requirement of ADAMTS-13 enzyme assays. Cost-effective, automated fiber-optic surface plasmon resonance (FO-SPR) platforms show potential for developing diagnostic tests. Yet, FO-SPR has never been explored to measure enzymatic activities.ObjectivesTo develop an easy-to-use ADAMTS-13 activity assay utilizing optical fibers to rapidly diagnose TTP.MethodsThe ADAMTS-13 activity assay was designed and optimized using FO-SPR technology based on a previously described enzyme-linked immunosorbent assay setup. A calibration curve was generated to quantify ADAMTS-13 activity in plasma of healthy donors and patients with acute immune-mediated TTP (iTTP), hemolytic uremic syndrome, or sepsis. ADAMTS-13 activity data from FO-SPR and fluorescence resonance energy transfer-based strategies (FRETS)-VWF73 reference assays were compared.ResultsAfter initial assay development, optimization improved read-out magnitude and signal-to-noise ratio and reduced variation. Further characterization demonstrated a detection limit (6.8%) and inter-assay variation (Coefficient of variation, 7.2%) that showed good analytical sensitivity and repeatability. From diverse plasma samples, only plasma from patients with acute iTTP showed ADAMTS-13 activities below 10%. Strong Pearson correlation (r = 0.854) between FO-SPR and reference FRETS-VWF73 assays were observed for all measured samples.ConclusionsA fast ADAMTS-13 activity assay was designed onto automated FO-SPR technology. Optimization resulted in sensitive ADAMTS-13 activity measurements with a detection limit enabling clinical diagnosis of TTP within 3 hours. The FO-SPR assay proved strong correlation with the reference FRETS-VWF73 assay. For the first time, this assay demonstrated the capacity of FO-SPR technology to measure enzymatic activity in pre-clinical context.

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

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