Gabriel M F, Uriel N, Teifoori F, Postigo I, Suñén E, Martínez J
Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, Laboratory of Parasitology and Allergy, Lascaray Research Centre, University of the Basque Country, Vitoria, Spain.
Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, Laboratory of Parasitology and Allergy, Lascaray Research Centre, University of the Basque Country, Vitoria, Spain.
Int J Food Microbiol. 2017 Sep 18;257:26-30. doi: 10.1016/j.ijfoodmicro.2017.06.006. Epub 2017 Jun 8.
The ubiquitously present spores of Alternaria alternata can spoil a wide variety of foodstuffs, including a variety of fruits belonging to the Citrus genus. The major allergenic protein of A. alternata, Alt a 1, is a species-specific molecular marker that has been strongly associated with allergenicity and phytopathogenicity of this fungal species. This study aimed to evaluate the potential of the detection of Alt a 1 as a reliable indicator of A. alternata contamination in citrus fruits. To accomplish this aim, sixty oranges were artificially infected with a spore suspension of A. alternata. Internal fruit material was collected at different incubation times (one, two and three weeks after the fungal inoculation) and used for both total RNA extraction and protein extraction. Alt a 1 detection was then performed by polymerase chain reaction (PCR) amplification using Alt a 1 specific primers and by enzyme-linked immunosorbent assay (ELISA). The experimental model presented in this work was effective to simulate the typical Alternaria black rot phenotype and its progression. Although both PCR and ELISA techniques have been successfully carried out for detecting Alt a 1 allergen in A. alternata infected oranges, the PCR method was found to be more sensitive than ELISA. Nevertheless, ELISA results were highly valuable to demonstrate that considerable amounts of Alt a 1 are produced during A. alternata fruit infection process, corroborating the recently proposed hypothesis that this protein plays a role in the pathogenicity and virulence of Alternaria species. Such evidence suggests that the detection of Alt a 1 by PCR-based assay may be used as a specific indicator of the presence of pathogenic and allergenic fungal species, A. alternata, in fruits. This knowledge can be employed to control the fungal infection and mitigate agricultural losses as well as human exposure to A. alternata allergens and toxins.
链格孢无处不在的孢子会破坏多种食品，包括柑橘属的各种水果。链格孢的主要致敏蛋白Alt a 1是一种物种特异性分子标记，与该真菌物种的致敏性和植物致病性密切相关。本研究旨在评估检测Alt a 1作为柑橘类水果中链格孢污染可靠指标的潜力。为实现这一目标，用链格孢的孢子悬浮液人工感染了60个橙子。在不同的培养时间（真菌接种后1、2和3周）收集果实内部材料，用于总RNA提取和蛋白质提取。然后使用Alt a 1特异性引物通过聚合酶链反应（PCR）扩增和酶联免疫吸附测定（ELISA）进行Alt a 1检测。本研究中提出的实验模型有效地模拟了典型的链格孢黑腐表型及其发展过程。虽然PCR和ELISA技术都已成功用于检测感染链格孢的橙子中的Alt a 1过敏原，但发现PCR方法比ELISA更灵敏。然而，ELISA结果对于证明在链格孢感染果实过程中产生了大量的Alt a 1非常有价值，证实了最近提出的该蛋白在链格孢属物种的致病性和毒力中起作用的假设。这些证据表明，基于PCR的检测方法检测Alt a 1可作为水果中致病和致敏真菌物种链格孢存在的特异性指标。这一知识可用于控制真菌感染，减轻农业损失以及减少人类接触链格孢过敏原和毒素。
