贵州医科大学学报

2021, v.46;No.244(01) 62-66

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实时荧光环介导等温扩增快速检测耐甲氧西林金黄色葡萄球菌方法的建立与评价
Establishment and evaluation of real-time fluorescence loop-mediated isothermal amplification for detection of MRSA

赵峻英;董剑;何建春;梅小亿;欧国平;
ZHAO Junying;DONG Jian;HE Jianchun;MEI Xiaoyi;OU Guoping;Department of Clinical Laboratory,the People's Hospital of Dazu District;

摘要(Abstract):

目的建立实时荧光环介导恒温扩增(Rea-LAMP)快速检测耐甲氧西林金黄色葡萄球菌(MRSA)的方法。方法针对葡萄球菌的mec A耐药标志性基因和SA特有的fem A基因的6个区域设计6条特异性引物,对MRSA、MSSA以及其他菌株等19株标准菌株的全基因组DNA进行Rea-LAMP,评价这些标准菌株的特异性、灵敏度和重复性;根据这些标准菌株的特异性,用Rea-LAMP反应体系检测临床分离的68株金黄色葡萄球菌(SA)菌株,并与VITEK2 Compact全自动微生物分析仪鉴定结果进行比较。结果本研究体系的引物扩增效果好,无引物二聚体,在19株标准菌株的检测中,除了MRSA和MSSA有相应的反应曲线外,其余菌株均为阴性;本体系的灵敏度可达1μg/L,68株临床SA菌株检测结果与VITEK2 Compact全自动微生物分析仪鉴定结果一致。结论 Rea-LAMP反应体系检测MRSA特异性强、稳定性好、灵敏度高、操作简单快捷,可用于MRSA的快速检测。
Objective To establish the real-time fluorescence loop-mediated isothermal amplification( Rea-LAMP) for rapid detection of multiple-resistant staphylococcus aureus( MRSA). Method A set of six specially primers were designed based on mec A and fem A genes in 6 distinct sequences. The amplification was obtained by incubating all of the reagents in a single tube for about 60 min of thermostatic reaction with Bst DNA polymerase,and the real-time detection of MRSA was conducted through fluorescent signals. The genomic DNAs of 19 standard strains such as MRSA and MSSA were extracted for conducting Rea-LAMP. Then,specificity,the lowest detectable limit and stability were evaluated. 68 strains of Staphylococcus aureus were tested by Rea-LAMP and the results obtained by VITEK2 with Rea-LAMP were compared. Results The primer amplification effect is good,without primer dimer. The MRSA and MSSA had amplification reaction respectively. While the strains of nonMRSA or non-MSSA had no amplification reaction. The REA-LAMP detection limit could reach 1 μg/L.The detection results of 68 clinical strains were consistent with those of VITEK2 automatic microbiological analyzer. Conclusion The Rea-LAMP is highly specific,stable,and sensitive for the detection of MRSA,and its operation is simple and rapid,which can be used for the rapid detection of MRSA.

关键词(KeyWords): 耐甲氧西林金黄色葡萄球菌;实时荧光;环介导恒温扩增;mecA基因;femA基因
multiple-resistant staphylococcus aureus(MRSA);real-time fluorescence quantitative;loop-mediated isothermal amplification(LAMP);mecA gene;femA gene

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基金项目(Foundation): 重庆市科卫联合基金资助项目(2018MSXM059)

作者(Author): 赵峻英;董剑;何建春;梅小亿;欧国平;
ZHAO Junying;DONG Jian;HE Jianchun;MEI Xiaoyi;OU Guoping;Department of Clinical Laboratory,the People's Hospital of Dazu District;

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DOI: 10.19367/j.cnki.2096-8388.2021.01.011

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