Abstract：Objective To establish a high throughout detecting method for testing Staphylococcus by GeXP system using CY5-labeled peculiar primer instead of universal primer. Methods Primers for 16S rDNA of bacteria, methicillin resistant gene (mecA, MecA LGA251) and macrolide resistant gene (ermA, ermB, ermC, sat4, msrA, ereA, ereB, and mefA/E) were designed. 129 strains of methicillin resistant or macrolide resistant Staphylococcus isolates were tested by PCR, while PCR was optimized. The forward primers were marked by CY5 and mixed together for establishing multiplex PCR of GeXP system. The multiplex PCR was performed to test muti-resistant genes of Staphylococcus. PCR productions were analyzed by agarose gel electrophoresis and GeXP electrophoresis. In addition, PCR was optimized based on the analysis of electrophoresis results. More than 43 strains of Staphylococcus were tested by GeXP multiplex PCR for further evaluation. Results Of ten genes, 7 (16S rDNA, mecA, ermA, ermB, ermC, sat4, msrA) were positive from 129 strains of Staphylococcus. Primers of 16S rDNA, mecA, ermA, ermB, ermC, and msrA produced high specificity, and primers of sat4 could produce two sizes of amplification fragments from different isolates. The detection results of 43 strains of Staphylococcus are in accordance with the VITEKⅡ Compact System. The coincidence rates between agarose gel electrophoresis and GeXP electrophoresis were 100% (43/43) for ermA, mecA, 16S rRNA, ermB and ermC, 97.67% (42/43) for sat4 (P＞0.05), and 95.35% (41/43) for msrA (P＞0.05). There was no significant statistical difference. Conclusion Labeled peculiar primer could work effectively in GeXP system for detecting multi-resistant genes of Staphylococcus, which produced high specificity and susceptibility. Additionally, its detection throughout could be higher if more primers were applied.
梁有才1 范菲楠2 聂署萍2 吴润香2 黄烈2 陆学东2,*. 引物标记技术的应用与葡萄球菌多重耐药基因检测法的建立[J]. 中国抗生素杂志, 2018, 43(6): 722-728.
Liang You-cai1, Fan Fei-nan2, Nie Shu-ping2, Wu Run-xiang2 , Huang Lie2 and Lu Xue-dong2. Application of labeled peculiar primer for detection of multidrug resistance Staphylococcus. CJA, 2018, 43(6): 722-728.