基因分型<em>金黄色葡萄球菌</em>由核糖体垫片的PCR（RS-PCR）
Summary
Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.
Abstract
The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.
Introduction
葡萄球菌 （ 金黄色葡萄球菌 ）被称为全球用于在牛1乳房内感染（IMI）负责的最重要的病原体。在12个欧洲国家的奶牛研究由Fournier 等人 2瑞士奶牛示范和研究通过Cosandey 等 3和老板等 4 S.金黄色葡萄球菌从牛隔离IMI是一种遗传异质组。由16S-23S rRNA基因间区（RS-PCR）的PCR扩增，共有17个基因型在101流行病学独立的分离2最初检测。基因型B和C型（GTC）是最常见的（80％），而其它15个基因型（GTS）发生很少，各使所有的菌株1至4％。在欧洲3级，GTB，GTC，GTF，GTI和GTR是由所有分析的株（N = 456）的76％，最重要的基因型。 GTB是位于欧洲中部W¯¯hereas其他基因型广泛传播。菌株的其余24％包含41种基因型，其中许多，但是，观察到只有一次。
通过Fournier 等人的 2，Cosandey 等 3和格雷伯等人 5的研究进一步表明，基因型与它们的毒力基因图案在瑞士高度相关的，以及如在欧洲一级。该研究进一步揭示之间S.在IMI患病率差异巨大的金黄色葡萄球菌 GTB，GTC和其他基因型2,5：考虑GTB，最多在牛群奶牛的87％是由这种基因型感染。在GTC和其他基因型的情况下，然而，IMI仅在1至2的奶牛畜群的发现。
IMI引起S.黄色葡萄球菌 ，通常会导致在相应的乳腺炎症和牛奶中的增加的炎性细胞。因此，体细胞共牛奶UNTS（SCC），牛奶的品质在大多数国家的关键指标，增加从而导致降低牛奶价格甚至停止发货。
除了Fournier 等人 2的RS-PCR其他分型方法已为亚型S的牛菌株描述金黄色葡萄球菌 8-11。两种最常见的包括水疗打字12多位点序列分型（MLST）13。前者是基于DNA测序葡萄球菌温泉基因编码蛋白质A的可变间隔区，而后者要求7管家基因的测序。这意味着一个12和七个PCR的13，分别需要执行，随后扩增子纯化，测序反应和分析使用特定的设备。相比的RS-PCR，这些方法需要在导致低的样品通量和成本高的实验室一个相当大的额外的努力。该吞吐量是特别低PFGE。考虑到这些方法对S的牛株分辨率金黄色葡萄球菌 ，RS-PCR是至少作为温泉打字4和优于MLST 4或电泳15一样好。
所有这些方法包括二进制打字13表现在获得进一步的深入了解造成S.牛IMI的发病机制及其有效性所提到的限制的金黄色葡萄球菌 ，但因为它们是用于大型临床研究较少为宜。另外，作为由Fournier 等人描述由RS-PCR基因分型。2允许流行病学和S的致病潜力的可靠的预测金黄色葡萄球菌参与牛IMI，在临床兽医学两个关键值。
Protocol
Representative Results
Discussion
整个过程中最关键的步骤是当在RS-PCR（> 30纳克纯的DNA /测定）过量的DNA的2。在一般情况下，一个轮廓的分辨率为最佳，如果所有其峰在基线开始和结束。如果两个峰值靠得太近，分辨率是不完整的。在这些条件下，从而需要人工识别的生物分析仪软件可能导致不适当的峰鉴定。
所有表现为BP或FU的明显分离，相关峰作进一步的分析。太多模板DNA RS-PCR结果在宽峰，因…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The author thanks R. Boss, A. Cosandey, C. Fournier, I. Ivanovic, and J. Naskova for their excellent work.
Materials
| Tris(hydroxymethyl)-aminomethan | Merck | 1.08382.0500 | Mw =121.14g/mol |
| Titriplex III | Merck | 1.08418.0250 | Mw = 372.24g/mol; Substance is equivalent to Na2EDTA·2H2O |
| Hydrochloric acid 5mol/l | Merck | 1.09911.0001 | |
| Columbia agar+5% sheep blood | bioMérieux | 43049 | |
| Agilent DNA7500 Kit | Agilent Technologies | 5067-1504 | |
| Agilent 2100 Bioanalyzer | Agilent Technologies | G2940CA | |
| Agilent 2100 expert sofware | Agilent Technologies | ||
| HotStarTaq master mix | Qiagen | 203445 | |
| Primer L1 | Mycrosinth | 5'CAA GGC ATC CAC CGT3' | |
| Primer G1 | Mycrosinth | 5'GAA GTC GTA ACA AGG3' |
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