脉冲场凝胶电泳(PFGE)是参考和临床实验室中用于分型耐甲氧西林金黄色葡萄球菌(MRSA)的最常见的基因型方法。在具有丰富的技术经验并建立了国家数据库的实验室中已经开发了许多不同的协议。然而,针对MRSA的不同欧洲PFGE方案和各种国家MRSA克隆本身的可比性至今尚未得到解决.这个跨国欧盟(EU)项目首次建立了欧洲代表性流行MRSA(EMRSA)菌株的数据库,并通过使用由共识方法开发的新的“协调”PFGE方案对它们进行了比较,该方案已证明具有足够的可重复性,可以在实验室之间成功比较脉冲场凝胶并跟踪欧盟周围的菌株。每个中心将来自八个欧洲国家的10个实验室的内部协议与“黄金标准”或初始协调协议进行了比较,其中许多参数已标准化。该小组发现标准化协议的某些要素并不重要,比如琼脂糖的类型,DNA块制备,和消化塞。其他要素被证明是至关重要的,即,琼脂糖的标准凝胶体积和浓度,插头中的DNA浓度,使用的离子强度和运行缓冲液的体积,运行温度,电压,和电泳的切换次数。商定了一项新的统一议定书,在两个实验室的试点研究中进一步修改,最后被所有其他人测试。发现七个实验室的凝胶具有足够好的质量,可以使用计算机软件程序对菌株进行比较。而由于不充分的脱色和DNA过载,两种凝胶无法分析。在尝试中心间PFGE比较之前,高质量的凝胶和内部质量控制菌株的包含是必不可少的。已显示许多克隆相关菌株存在于整个欧洲的多个国家中。著名的伊比利亚克隆已经在比利时得到证明,芬兰,法国,德国,和西班牙(以及葡萄牙更广泛的HARMONY系列,斯洛文尼亚,和瑞典)。来自英国的菌株(EMRSA-15和-16)已经在其他几个国家被确认,和其他克隆相关菌株也已被鉴定。这突出了需要更密切的国际合作,以监测当前流行菌株的传播以及新菌株的出现。
Pulsed-fieldgel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new \"harmonized\" PFGE protocol developed by a
consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a \"gold standard\" or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories\' gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium, Finland, France, Germany, and Spain (and from the wider HARMONY collection in Portugal, Slovenia, and Sweden). Strains from the United Kingdom (EMRSA-15 and -16) have been identified in several othercountries, and other clonally related strains have also been identified. This highlights the need for closer international collaboration to monitor the spread of current epidemic strains as well as the emergence of new ones.