Institute of Applied Biosciences, CERTH, Thessaloniki, Greece.
Institute of Plant Breeding and Genetic Resources ELGO-DEMETER, Thessaloniki, Greece.
J Sci Food Agric. 2019 Jan 15;99(1):309-314. doi: 10.1002/jsfa.9189. Epub 2018 Jul 17.
Aspergillus is a diverse genus of fungi with high economic and social impact. Various species that belong to section Nigri (black aspergilli) are common agents of grape spoilage and potent producers of ochratoxin A (OTA), a mycotoxin associated with various nephrotoxic and immunotoxic effects in humans. Black aspergilli are difficult to classify following only phenotypic criteria; thus chemotaxonomic and molecular methods are employed in parallel with phenotypic ones for species characterization. These approaches, though accurate and replicable, require more than one individual step and are to a certain extent laborious when a rapid identification of these species is required.
The aim of this study was to develop a high-resolution melting polymerase chain reaction (HRM-PCR) assay as a rapid method for identification of Aspergillus spp. section Nigri isolates and their detection in grape samples. Melt curve analysis of amplicons originating from the internal transcribed spacer 2 (ITS2) ribosomal region generated species-specific HRM curve profiles, enabling the accurate differentiation of the analyzed genotypes. Furthermore, the assay was able to identify A. carbonarius, A. tubingensis, A. niger, A. ibericus and A. japonicus in grape samples artificially inoculated with conidia of these fungi.
To our knowledge this is the first report on the development of an HRM-PCR assay for the identification of black Aspergillus species in grape samples. © 2018 Society of Chemical Industry.
曲霉菌是一个具有高度经济和社会影响的真菌多样性属。属于 Nigri 组（黑曲霉）的各种物种是葡萄腐烂的常见原因，也是赭曲霉毒素 A（OTA）的强有力生产者，OTA 是一种与人类各种肾毒性和免疫毒性有关的霉菌毒素。仅根据表型标准很难对黑曲霉进行分类；因此，在对物种进行特征描述时，会同时采用化学分类学和分子方法以及表型方法。这些方法虽然准确且可重复，但需要多个单独的步骤，并且在需要快速识别这些物种时在一定程度上很繁琐。
本研究旨在开发一种高分辨率熔解聚合酶链反应（HRM-PCR）检测方法，作为一种快速鉴定 Aspergillus 属 Nigri 组分离株及其在葡萄样品中检测的方法。来自核糖体内部转录间隔区 2（ITS2）的扩增子的熔解曲线分析产生了物种特异性的 HRM 曲线谱，能够准确区分分析的基因型。此外，该检测方法能够鉴定出人工接种这些真菌分生孢子的葡萄样品中的 A. carbonarius、A. tubingensis、A. niger、A. ibericus 和 A. japonicus。
据我们所知，这是首次报道用于鉴定葡萄样品中黑曲霉物种的 HRM-PCR 检测方法。 © 2018 化学工业协会。
