环介导等温扩增法快速检测耐甲氧西林金黄色葡萄球菌

摘要

建立一种应用环介导等温扩增（loop-mediated isothermal amplification,LAMP）方法快速检测耐甲氧西林金黄色葡萄球菌（methicillin-resistan Staphylococcus aureus,MRSA）中mecA和spa基因的检测方法。以金葡菌和其他相关菌种为试验对象,分别用聚合酶链式反应（ploymerase chain reaction,PCR）和LAMP方法检测mecA和spa基因。结果表明：LAMP法在64℃等温条件下可在60min内成功扩增基因,且与传统PCR方法结果相同;通过琼脂糖凝胶电泳可知,LAMP对mecA和spa基因的检测限分别为每菌管102个和10个细胞;肉眼可检测到的mecA和spa基因的检测限分别为每菌管103个和10个细胞;然后用LAMP法检测人工污染原料乳样本中MRSA,用LAMP法检测原料乳中的mecA和spa基因与PCR方法显示出相同的结果。LAMP方法可快速检测mecA和spa基因,此方法可应用于原料乳中MRSA的检测。%A rapid assay was developed for the detection of mecA and spa gene present in methicillin-resistant Staphylococcus aureus（MRSA） using loop-mediated isothermal amplification（LAMP）.Staphylococcus aureus and other relevant bacteria were detected for their mecA and spa genes by both polymerase chain reaction（PCR） and LAMP methods.Gene amplification was successfully achieved by LAMP within 60 min when the temperature was held at 64 ℃,and the results were consistent results with those of the conventional PCR methods.The limits of detection of the LAMP method for mecA and spa genes were identified by agarose gel electrophoresis to be 102 and 10 cells per tube and observed by naked eyes to be 103 and 10 cells per tube respectively.The LAMP method was then applied to artificially polluted raw milk,and gave identical results with the traditional PCR methods.LAMP can rapidly detect mecA and spa genes,thus providing an applicable assay for detecting MRSA in raw milk.