Selma María V, Martínez-Culebras Pedro V, Aznar Rosa
Department of Microbiology and Ecology, University of Valencia, Burjassot E-46100, Valencia, Spain.
Int J Food Microbiol. 2008 Feb 29;122(1-2):126-34. doi: 10.1016/j.ijfoodmicro.2007.11.049. Epub 2007 Nov 29.
Aspergillus carbonarius is the main species responsible for ochratoxin A accumulation in wine grapes and consequently, its rapid and sensitive detection is increasingly investigated. A new real-time PCR (RTi-PCR) based procedure was developed for the rapid and specific detection and quantification of A. carbonarius in wine grapes. The procedure includes the use of the pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors, and DNA extraction with the EZNA Fungal DNA kit. It reduced the time for A. carbonarius DNA extraction from grapes to 30 min. Two specific primers (AcKS10L/AcKS10R) delimiting a 161 bp fragment, and a probe were designed and directed to the beta-ketosynthase domain of a polyketide synthase from A. carbonarius. Specificity was confirmed by testing primers towards purified DNA from 52 fungal strains, including reference and food isolates. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated conidial suspensions from A. carbonarius. The SYBR-Green I and TaqMan RTi-PCR approaches established were able to detect at least 2.4 and 24 genomic equivalents, respectively, using purified DNA. Results obtained from conidial suspensions, after DNA extraction, showed that at least 5 conidia per reaction should be present for a positive result with SYBR-Green I and 50 in the case of TaqMan. The quantification of fungal genomic DNA in artificially inoculated wine grapes performed successfully, with a minimum threshold of 10(3) conidia mL(-1) for accurate quantification. The developed RTi-PCR assay is a promising tool in the prediction of potential ochratoxigenic risk, even in the case of low-level infections, and suitable for a rapid, automated and high throughput analysis.
黑曲霉是导致酿酒葡萄中赭曲霉毒素A积累的主要菌种，因此，对其进行快速灵敏的检测受到了越来越多的研究。一种基于实时荧光定量PCR（RTi-PCR）的新方法被开发出来，用于快速、特异性地检测和定量酿酒葡萄中的黑曲霉。该方法包括使用脉动器设备去除葡萄上的分生孢子，以防止PCR抑制剂的释放，并使用EZNA真菌DNA试剂盒进行DNA提取。它将从葡萄中提取黑曲霉DNA的时间缩短至30分钟。设计了两条特异性引物（AcKS10L/AcKS10R），它们界定了一个161bp的片段，并设计了一个探针，靶向黑曲霉聚酮合酶的β-酮合成酶结构域。通过用52种真菌菌株（包括参考菌株和食品分离株）的纯化DNA测试引物，确认了特异性。使用纯化DNA的系列稀释液和黑曲霉校准的分生孢子悬浮液，定量在至少5个对数单位内呈线性。所建立的SYBR-Green I和TaqMan RTi-PCR方法分别使用纯化DNA能够检测到至少2.4个和24个基因组当量。DNA提取后从分生孢子悬浮液获得的结果表明，对于SYBR-Green I，每个反应至少应有5个分生孢子才能得到阳性结果，对于TaqMan则为50个。在人工接种的酿酒葡萄中成功地进行了真菌基因组DNA的定量，准确量化的最低阈值为10³个分生孢子/mL。所开发的RTi-PCR检测方法是预测潜在赭曲霉毒素产生风险的一种有前途的工具，即使在低水平感染的情况下也是如此，并且适用于快速、自动化和高通量分析。
