Cruz Patricia, Buttner Mark P
Microbiology Division, Harry Reid Center for Environmental Studies, University of Nevada, Las Vegas, 4505 S. Maryland Parkway, Box 4009, Las Vegas, Nevada 89154-4009, USA.
Mycologia. 2008 Sep-Oct;100(5):683-90. doi: 10.3852/08-022.
Aspergillus flavus is a ubiquitous mold and the most common mold contaminating foodstuffs. Many strains of A. flavus produce aflatoxins. In addition it is an allergen and an opportunistic pathogen of animals and plants. A. flavus often is underestimated in traditional culture analyses due to the expertise required and the cost associated with speciating members of the genus Aspergillus. The goal of this study was to develop and validate a primer and probe set for the rapid detection and quantitation of A. flavus in pure culture using real-time quantitative polymerase chain reaction (QPCR) amplification. Unique DNA regions were located in the genome of the target organism by sequence comparison with the GenBank database, and several candidate oligonucleotides were identified from the scientific literature for potential use with the TaqMan QPCR technology. Three primer and probe sets were designed and validated for specificity and sensitivity in laboratory experiments. Initial screening to test for sensitivity was performed with seven A. flavus isolates and selected nontarget fungi. Specificity testing was conducted with the selected primer and probe set, which amplified all nine A. flavus isolates tested, including an aflatoxin producing strain. The primers did not amplify DNA extracted from 39 other fungal species (comprising 16 genera), including 18 other Aspergillus species and six Penicillium species. No amplification of human or bacterial DNA was observed; however cross-reactivity was observed with Aspergillus oryzae. PCR analysis of DNA dilutions and the use of an internal positive control demonstrated that 67% of the fungal DNA samples assayed contained PCR inhibitors. The assay validated for the target organism is capable of producing PCR results in less than 1 h after DNA extraction. The results of this research demonstrate the capabilities of QPCR for the enhanced detection and enumeration of fungi of significance to human health.
黄曲霉是一种广泛存在的霉菌，也是污染食品最常见的霉菌。许多黄曲霉菌株会产生黄曲霉毒素。此外，它还是一种过敏原，是动植物的机会性病原菌。由于鉴定曲霉属成员需要专业知识且成本较高，黄曲霉在传统培养分析中常常被低估。本研究的目的是开发并验证一套引物和探针，用于通过实时定量聚合酶链反应（QPCR）扩增对纯培养物中的黄曲霉进行快速检测和定量。通过与GenBank数据库进行序列比较，在目标生物体的基因组中定位了独特的DNA区域，并从科学文献中鉴定出几种候选寡核苷酸，用于TaqMan QPCR技术。设计并验证了三套引物和探针在实验室实验中的特异性和灵敏度。最初的敏感性筛选是用七株黄曲霉分离株和选定的非靶标真菌进行的。使用选定的引物和探针组进行特异性测试，该引物和探针组扩增了所有测试的九株黄曲霉分离株，包括一株产黄曲霉毒素的菌株。这些引物未扩增从39种其他真菌物种（包括16个属）中提取的DNA，其中包括18种其他曲霉菌种和6种青霉菌种。未观察到人类或细菌DNA的扩增；然而，观察到与米曲霉有交叉反应。对DNA稀释液的PCR分析以及使用内部阳性对照表明，所检测的真菌DNA样本中有67%含有PCR抑制剂。针对目标生物体验证的检测方法能够在DNA提取后不到1小时产生PCR结果。本研究结果证明了QPCR在增强检测和计数对人类健康具有重要意义的真菌方面的能力。
