Department of Microbiology and Ecology, University of Valencia, Valencia, Spain.
Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2009 Feb;26(2):180-8. doi: 10.1080/02652030802345623.
This paper reports a duplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of members of the Aspergillus niger aggregate and A. carbonarius, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the poliketide synthase of A. carbonarius and the A. niger aggregate, respectively. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic T(m)-values demonstrating the specific, efficient and balanced amplification of the two PCR fragments. Subsequently, a TaqMan real-time PCR approach was settled, using 6-carboxy-fluorescein group (FAM) and VIC-labelled specific probes for automated detection. Results indicated no differences in sensitivity when using either the two sets of primers and probes in separate or in the same reaction. However, when both targets are in very different amounts, there is a preferential amplification of the target which is in more concentration. CT-values obtained in the presence of grape DNA were very similar to those observed when only fungal purified DNA was present, indicating that the grape DNA does not interfere in the real-time PCR reaction. This procedure provides a fast and accurate tool to monitor, in a single reaction, the presence of OTA-producing species in grapes which, to some extent, will facilitate OTA contamination surveys to guarantee food safety in the wine industry.
本文报道了一种用于同时检测黑曲霉聚集体和 A. carbonarius 的双重实时聚合酶链反应 (PCR) 检测方法，这两种菌是葡萄中赭曲霉毒素 A (OTA) 污染的主要责任菌。该单管反应针对 A. carbonarius 和 A. niger 聚集体的聚酮合酶的β-酮基合成酶和酰基转移酶结构域。使用 SYBR Green I 实时 PCR 方法进行的熔解曲线分析显示出特征 T(m) 值，证明了两个 PCR 片段的特异性、高效和平衡扩增。随后，建立了 TaqMan 实时 PCR 方法，使用 6-羧基-荧光素基团 (FAM) 和 VIC 标记的特异性探针进行自动检测。结果表明，在单独或同一反应中使用两组引物和探针时，灵敏度没有差异。然而，当两个靶标在数量上非常不同时，存在优先扩增浓度更高的靶标。在存在葡萄 DNA 的情况下获得的 CT 值与仅存在真菌纯化 DNA 时观察到的 CT 值非常相似，表明葡萄 DNA 不会干扰实时 PCR 反应。该程序提供了一种快速准确的工具，可在单次反应中监测葡萄中产生 OTA 的物种的存在，这在一定程度上有助于进行 OTA 污染调查，以确保葡萄酒行业的食品安全。
