The monitoring of the fungal genus Alternaria, which causes destructive brown spot disease in citruses worldwide and produces highly toxic mycotoxins, is extremely important to protect citrus and human health. In this work, we describe an ultrasensitive colorimetric method for the detection of genomic DNA of Alternaria from citrus fruit samples, using a system consisting of five groups of reporter probes. Each reporter probe is prepared by coupling recognition DNA and horseradish peroxidase (HRP) on the surface of gold nanoparticle (AuNP) through a convenient and low-cost freezing-assisted method. Meanwhile, the capture DNA is immobilized on magnetic bead (MB) via biotin-streptavidin reaction. Then, the capture DNA, target DNA, and five groups of AuNP-based reporter probes form a stable DNA-heptamer sandwich structure on the MB, and then HRP generates a blue signal for the subsequent colorimetric detection. It should be noted that AuNP with a large specific surface area drives abundant HRP anchoring, resulting in significant signal amplification. In addition, there are five groups of AuNP-based reporter probes, which further amplify the detection signal. As a result, the detection limit of the artificial target DNA is as low as 15.6 pM. Because the detection signal can be recorded visually without any special equipment, and its sensitivity is high, this method represents a suitable diagnostic tool for Alternaria genetic detection.
Keywords: Alternaria; Citrus; Colorimetric DNA biosensor; Gold nanoparticle; Horseradish peroxidase.
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