Lin Qun, Xu Pusheng, Li Jiaowu, Chen Yin, Feng Jieyi
Lecong Hospital Affiliated of Guangzhou Medical University, Shunde District, Foshan 528315, Guangdong province, China.
Lecong Hospital Affiliated of Guangzhou Medical University, Shunde District, Foshan 528315, Guangdong province, China.
Microb Pathog. 2017 Aug;109:183-188. doi: 10.1016/j.micpath.2017.05.044. Epub 2017 May 31.
Loop-mediated isothermal amplification based detection assays using bacterial culture or colony for direct detection of methicillin resistant Staphylococcus aureus(MRSA) had been developed and evaluated, followed by its extensive application on a large scale of clinical MRSA isolated from respiratory origins, including nasal swabs and sputums. Six primers, including outer primers, inner primers and loop primers, were specifically designed for recognizing eight distinct sequences on four targets: 16SrRNA, femA, mecA and orfX. Twenty-seven reference strains were used to develop, evaluate and optimize this assay. Then, a total of 532 clinical MRSA isolates were employed for each detected targets. And the results were determined through both visual observation of the color change by naked eye and electrophoresis. The specific of each primer had been confirmed, and the optimal amplification was obtained under 65 °C for 40 min. The limit of detections (LOD) of bacteria culture LAMP assays were determined to be 10 CFU/ml for 16S rRNA, femA, as well as orfX and 10 CFU/ml for mecA, respectively. The established novel assays on MRSA detection may provide new strategies for rapid detection of foodborne pathogens.
已经开发并评估了基于环介导等温扩增的检测方法，该方法使用细菌培养物或菌落直接检测耐甲氧西林金黄色葡萄球菌（MRSA），随后将其广泛应用于从呼吸道来源分离的大量临床MRSA，包括鼻拭子和痰液。专门设计了六种引物，包括外引物、内引物和环引物，用于识别四个靶标（16SrRNA、femA、mecA和orfX）上的八个不同序列。使用27株参考菌株来开发、评估和优化该检测方法。然后，对每个检测靶标使用总共532株临床MRSA分离株。结果通过肉眼观察颜色变化和电泳来确定。已确认每种引物的特异性，并在65°C下40分钟获得最佳扩增效果。细菌培养LAMP检测方法对16S rRNA、femA以及orfX的检测限分别确定为10 CFU/ml，对mecA的检测限为10 CFU/ml。所建立的新型MRSA检测方法可能为食源性病原体的快速检测提供新策略。
