Instituto de Recursos Naturales y Agrobiología de Sevilla, IRNAS-CSIC, Apartado 1052, 41080 Sevilla, Spain.
Sci Total Environ. 2013 Jan 15;443:478-84. doi: 10.1016/j.scitotenv.2012.11.026. Epub 2012 Dec 4.
A real-time Polymerase Chain Reaction (PCR) assay was developed to detect and quantify Ochroconis lascauxensis in the Lascaux Cave in France. This fungus is the principal causal agent of the black stains threatening the Paleolithic paintings of this UNESCO World Heritage Site. The black stains outbreak could not be stopped in spite of using intensive biocide treatments. A sensitive and time-saving protocol is needed for determining the extent of the colonization. Sets of primers that target the ITS and RPB2 regions were designed and evaluated for specificity against O. lascauxensis. Genomic DNA extracted from five species of Ochroconis and 13 other fungal species frequently isolated from caves were used to test the specificity of each primer set. The specific and sensitive real-time PCR assay using the primers 347F/493R targeting a 147-bp fragment from the RPB2 gene was useful for quantifying the presence of O. lascauxensis in the stains on the walls, sediments and air of the cavity. The results confirmed the association of this fungus with the black stains and its wide dissemination in all cave compartments. The suitability of this method for monitoring fungal outbreaks in cave environments is discussed.
本研究开发了一种实时聚合酶链反应（PCR）检测方法，用于检测和定量法国拉斯科洞穴中的拉氏奥克戎菌。这种真菌是威胁联合国教科文组织世界遗产地洞穴内史前绘画的黑色污渍的主要致病因子。尽管采用了强化杀菌剂处理，黑色污渍的爆发仍无法得到控制。因此，需要一种敏感且省时的方法来确定污染的程度。本研究设计并评估了针对 ITS 和 RPB2 区域的引物对，以检测其对拉氏奥克戎菌的特异性。从 5 种奥克戎菌物种和 13 种其他经常从洞穴中分离出的真菌物种中提取基因组 DNA，用于测试每组引物的特异性。使用针对 RPB2 基因 147bp 片段的引物 347F/493R 进行的特异性和敏感的实时 PCR 检测，可用于定量检测壁上、沉积物中和洞穴空气中的拉氏奥克戎菌的存在。结果证实了该真菌与黑色污渍的关联性及其在洞穴所有区域的广泛传播。本文讨论了该方法在监测洞穴环境中真菌爆发的适用性。
