Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Simultaneous Detection of mecA, nuc, and femB by Loop-Mediated Isothermal Amplification (LAMP)
Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Simultaneous Detection of mecA, nuc, and femB by Loop-Mediated Isothermal Amplification (LAMP)
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通过环介导等温扩增 (LAMP) 同时检测 mecA、nuc 和 femB 来鉴定耐甲氧西林金黄色葡萄球菌 (MRSA)
DOI：
10.1007/s00284-017-1274-2
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发表时间：
2017-08-01
影响因子：
2.6
通讯作者：
Chen, Qiuyuan
中科院分区：
文献类型：
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作者：
Chen, Changguo;Zhao, Qiangyuan;Chen, Qiuyuan
本研究的目的是开发一种快速检测方法，通过使用环介导等温扩增（LAMP）方法同时检测mecA，nuc和femB基因来鉴定耐甲氧西林金黄色葡萄球菌。使用在线生物软件（http://primerexplorer.jp/e/）设计LAMP引物，并在等温浴中进行扩增反应。然后使用2%琼脂糖凝胶电泳检测产物。通过三重TaqMan实时PCR确认MecA、nuc和femB。为了更好地肉眼观察反应结果，在扩增体系中加入了羟基萘酚蓝（HNB）。在60 min内，LAMP在63 °C等温条件下成功扩增了目的基因。2%凝胶电泳结果表明，当反应体系中Mg2+浓度为6 μmol时，mecA基因的扩增效果较好，而nuc和femB基因的扩增效果较好，Mg2+浓度为8 μmol。向25 μL反应体系中加入1 μL（3.75 mM）HNB，观察到明显的颜色差异。应用LAMP法对日常标本中分离的128株耐甲氧西林金黄色葡萄球菌进行鉴定。LAMP和PCR的结果是相同的。LAMP为mecA、nuc和femB提供了另一种检测方法，并且比其他方法更快。
The aim of this study was to develop a rapid detection assay to identify methicillin-resistant Staphylococcus aureus by simultaneous testing for the mecA, nuc, and femB genes using the loop-mediated isothermal amplification (LAMP) method. LAMP primers were designed using online bio-software ( http://primerexplorer.jp/e/ ), and amplification reactions were performed in an isothermal temperature bath. The products were then examined using 2% agarose gel electrophoresis. MecA, nuc, and femB were confirmed by triplex TaqMan real-time PCR. For better naked-eye inspection of the reaction result, hydroxy naphthol blue (HNB) was added to the amplification system. Within 60 min, LAMP successfully amplified the genes of interest under isothermal conditions at 63 °C. The results of 2% gel electrophoresis indicated that when the Mg2+concentration in the reaction system was 6 μmol, the amplification of the mecA gene was relatively good, while the amplification of the nuc and femB genes was better at an Mg2+concentration of 8 μmol. Obvious color differences were observed by adding 1 μL (3.75 mM) of HNB into 25 μL reaction system. The LAMP assay was applied to 128 isolates cases of methicillin-resistant Staphylococcus aureus, which were separated from the daily specimens and identified by Vitek microbial identification instruments. The results were identical for both LAMP and PCR. LAMP offers an alternative detection assay for mecA, nuc, and femB and is faster than other methods.