The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbia1 ecology of the alteration process
The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbia1 ecology of the alteration process
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DOI：
10.1016/s0168-1605(03)00296-4
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发表时间：
2004-01-01
影响因子：
5.4
通讯作者：
Comi, G
中科院分区：
文献类型：
--
作者：
Cocolin, L;Innocente, N;Comi, G
开发了一种基于聚合酶链反应（PCR）和变性梯度凝胶电泳（DGGE）的分子生物学方法来检测梭菌属。疑似晚吹的奶酪样品中。梭菌属菌株。并使用从国际保藏中获得的不同乳酸菌物种来确定 PCR 扩增和 DGGE 分化的实验条件。直接从具有晚期发泡症状的奶酪中提取的 DNA 进行 PCR 和 DGGE 分析，并对巴氏灭菌和浓缩过夜的样品进行传统的琼脂平板接种。此外，为了比较目的还测定了挥发性脂肪酸。 PCR-DGGE 结果与进行的铺板一致，并且仅呈现 DGGE 带迁移到与梭菌属相同位置的样品。条带显示在强化梭菌培养基平板上存在梭菌菌落。在 DGGE 结果呈阳性的情况下，丁酸含量较高（>100 mg/kg），强调了该方案对于奶酪腐败研究的适用性。该方法的灵敏度估计为 10(4) CFU/g。 (C) 2003 Elsevier B.V. 保留所有权利。
A molecular biology method based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) was developed to detect Clostridium spp. in cheese samples suspected of late blowing. Strains of Clostridium spp. and different Lactic Acid Bacteria species, obtained from international collections, were used to determine the experimental conditions for the PCR amplification and DGGE differentiation. DNA extracted directly from cheeses with late blowing symptoms was subjected to PCR and DGGE analysis and traditional agar plating was performed for samples pasteurized and enriched overnight. Moreover, volatile fatty acids were determined for comparison purposes. The PCR-DGGE results were in agreement with the plating performed, and only samples presenting DGGE bands migrating at the same position as Clostridium spp. bands, showed the presence of Clostridium colonies on Reinforced Clostridial Medium plates. Butyric acid contents were high (>100 mg/kg) in the cases of positive DGGE results, underlining the suitability of the protocol for the study of cheese spoilage. The sensitivity of the method is estimated to be 10(4) CFU/g. (C) 2003 Elsevier B.V. All rights reserved.