Biotecnologia Enológica, Dept. Bioquímica i Biotecnologia, Facultat d'Enologia, Universitat Rovira i Virgili, C/ Marcel·lí Domingo s/n, 43007 Tarragona, Spain.
Food Microbiol. 2013 Oct;36(1):30-9. doi: 10.1016/j.fm.2013.03.008. Epub 2013 Apr 13.
The identification and quantification of Acetobacter malorum and Acetobacter cerevisiae in wine and vinegar were performed using the Real-Time PCR (RT-PCR) with two TaqMan-MGB probes designed to amplify the internal transcribed spacer (ITS) region between the 16S-23S rRNA genes. The primers and probes were highly specific, with a detection limit of 10² cells/ml for both species, and the efficiency of the technique was >80%. The RT-PCR technique with these two new TaqMan-MGB probes, together with the five (Acetobacter aceti, Acetobacter pasteurianus, Gluconobacter oxydans, Gluconacetobacter hansenii and Gluconacetobacter europaeus) that are already available (Torija et al., 2010), were validated on known concentrations of Acetic Acid Bacteria (AAB) grown in glucose medium (GY) and in inoculated matrices of wine and vinegar. Furthermore, this technique was applied to evaluate the AAB population in real wine samples collected in the Canary Islands. PCR enrichment performed prior to RT-PCR increased the accuracy of quantification and produced results similar to those detected with SYBR-Green. In real wine samples, the total AAB enumeration ranged from 9 × 10² to 10⁶ cells/ml, and the seven AAB species tested were detected in more than one sample. However, AAB recovery on plates was poor; the isolates obtained on plates were A. malorum, G. oxydans, A. cerevisiae and A. pasteurianus species. RT-PCR with TaqMan-MGB probes is an accurate, specific and fast method for the identification and quantification of AAB species commonly found in wine and vinegar.
使用针对 16S-23S rRNA 基因之间的内部转录间隔区（ITS）设计的两个 TaqMan-MGB 探针的实时 PCR（RT-PCR）来鉴定和定量葡萄酒和醋中的异常酿酒酵母和酿酒酵母。这些引物和探针具有高度特异性，两种物种的检测限均为 10²细胞/ml，该技术的效率>80％。使用这两种新的 TaqMan-MGB 探针的 RT-PCR 技术，以及现有的五种（醋杆菌，巴氏醋杆菌，氧化葡萄糖酸杆菌，汉逊氏醋酸杆菌和欧洲醋酸杆菌）（Torija 等人，2010），在葡萄糖培养基（GY）中生长的已知浓度的醋酸菌（AAB）和接种的葡萄酒和醋基质中进行了验证。此外，该技术还用于评估在加那利群岛收集的实际葡萄酒样品中的 AAB 种群。在 RT-PCR 之前进行的 PCR 富集提高了定量的准确性，并产生了与 SYBR-Green 检测相似的结果。在实际葡萄酒样品中，总 AAB 计数范围为 9×10²至 10⁶细胞/ml，并且在所测试的七种 AAB 物种中，有超过一种样品中检测到了这些物种。但是，平板上的 AAB 回收率很差；在平板上获得的分离物是异常酿酒酵母，氧化葡萄糖酸杆菌，酿酒酵母和巴氏醋杆菌。使用 TaqMan-MGB 探针的 RT-PCR 是一种准确，特异和快速的方法，可用于鉴定和定量葡萄酒和醋中常见的 AAB 物种。
