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  

Application of labeled peculiar primer for detection of multidrug resistance Staphylococcus

作　　者：梁有才 范菲楠[2] 聂署萍[2] 吴润香[2] 黄烈 陆学东[2] Liang You-cai;Fan Fei-nan;Nie Shu-ping;Wu Run-xiang;Huang Lie;Lu Xue-dong(Department of Graduate School, Guangdong Medical University, Zhanjiang 524000;Department of Laboratory Medicine, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen 518033)

机构地区：[1]广东医科大学研究生学院,湛江524000 [2]中山大学附属第八医院检验医学部,深圳518033

出　　处：《中国抗生素杂志》2018年第6期722-728,共7页Chinese Journal of Antibiotics

摘　　要：目的基于多重基因遗传信息表达分析系统(Ge XP)技术平台,应用CY5(荧光染料)标记的特异引物代替通用引物,建立一种高通量的葡萄球菌多重耐药基因检测法。方法设计细菌16S rDNA、耐甲氧西林相关基因(mecA、MecA LGA251)与耐大环内酯类相关基因(ermA、ermB、ermC、sat4、msrA、ereA、ereB、mefA/E)的引物,经"PCR+琼脂糖电泳"筛查2014年1月—2016年12月中山大学附属第八医院129株耐甲氧西林或者耐红霉素葡萄球菌基因,同时优化PCR。用CY5标记上游引物5'端,制作Ge XP多重PCR引物。应用多重PCR扩增多重耐药基因核酸,产物经琼脂糖电泳与Ge XP毛细管电泳,分析电泳图优化PCR。应用优化后的Ge XP多重PCR扩增43株葡萄球菌,验证应用效果。结果 129株葡萄球菌筛出7种基因(16S rDNA、mecA、ermA、ermB、ermC、sat4和msrA)。16S rDNA、mecA、ermA、ermB、ermC和msrA引物特异性好,但不同核酸的sat4基因能扩增出两种不同大小片段。建立的Ge XP多重PCR能同时检出所有靶基因。43株葡萄球菌的检测结果与表型检测(VITEKⅡCompact System)相符;与"PCR+琼脂糖电泳"相比:sat4符合率为97.67%(42/43),无统计学差异(P>0.05);msrA符合率为95.35%(41/43),无统计学差异(P>0.05);ermA、mecA、16S rDNA、ermB和ermC符合率均为100%(43/43)。结论采用CY5标记特异引物应用于GeXP系统检测葡萄球菌多重耐药基因的方法可行,其特异性与敏感性高,若采用更多引物,其检测通量可进一步提高。Objective To establish a high throughout detecting method for testing Staphylococcus by GeXP system using CY5-1abeled 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 Often 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 VITEK II 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.

关 键 词：标记引物 多重基因分析系统 甲氧西林耐药 大环内酯类耐药 葡萄球菌 耐药基因 

分 类 号：R978.1[医药卫生—药品]