摘要
目的建立一种简便、快速、特异、灵敏的耐碳青霉烯类鲍曼不动杆菌(CRAB)OXA-23基因分子检测方法,并用于临床标本检测,以便了解产OXA-23酶CRAB的耐药分布状况。方法运用环介导等温扩增技术(LAMP),利用PrimerExplorer 4.0软件设计一套特异引物检测CRAB的OXA-23基因。通过优化LAMP检测方法的实验条件,应用SYBR Green I作为LAMP反应荧光显色物质,然后通过肉眼目测或电泳检测比较结果。同时,应用LAMP检测该院自2013年12月至2014年3月收集分离的41株多重耐药鲍曼不动杆菌。结果 CRAB的OXA-23基因菌株LAMP反应电泳产生了阶梯状条带,而其他不同菌株和阴性对照没有条带;向扩增产物中加入1μL SYBR GreenⅠ,CRAB的OXA-23基因菌株LAMP扩增产物的颜色由橙色变为绿色,而其他不同菌株和阴性对照仍为橙色;并检测出CRAB的OXA-23基因菌株纯培养物的灵敏度达到了5cfu/μL。对41株我院分离的多重耐药鲍曼不动杆菌进行LAMP检测,检出OXA-23基因32株,检出率为78.04%。结论该院分离的CRAB OXA-23基因的携带率较高,对常用抗菌药物有非常高的耐药率;本研究建立的LAMP技术检测CRAB OXA-23基因是一种简便、快速、特异、灵敏的耐药鲍曼不动杆菌检测方法,适于基层单位推广使用,对临床医生合理选择抗生素具有重要意义。
Objective To establish a simple,rapid,highly specific and sensitive molecular detection of carbapenem-resistance acinetobacter baumannii（CRAB）OXA-23 genes,and this method is used to detect the multiple drug-resistant acinetobacter baumannii in our hospital,and the purpose is to know the antibiotic resistance of CRAB OXA-23 genes.Methods The loop-mediated isothermal amplification（LAMP）was established for detection of the CRAB OXA-23 genes,and a set of specific primers were designed by special software,PrimerExplorer version 4.The LAMP assay was developed on using SYBR Green Ⅰ for fluorescent chromogenic reaction substances,improved through a series of optimization tests,and through macroscopic observation and electrophoresis test comparison results.At the same time,the application of LAMP was used to test 41 multiple drug-resistant acinetobacter baumanniis which were collected from December 2013 to March 2014 in our hospitalized patients.Results The ladder banding was produced in CRAB OXA-23 genes strains by the LAMP detection through electrophoresis test,however,no ladder banding was observed in the others.The color of the amplification product in genes strain CRAB OXA-23 changed from orange to green by adding 1μL SYBR GreenⅠ,however it was still orange in others.The sensitivity of the LAMP detection in pure cultrue was 5cfu/μL of the CRAB OXA-23 genes cells.Application of LAMP was used to separate multiple drug-resistant acinetobacter baumanniis from hospitalized patients,32 strains were tested in 41 strains,the positive rate was 78.04%.Conclusion Separation of the CRAB OXA-23 genes carry rate is higher in our hospital,and they have very high resistance of commonly used antibacterial drugs.The LAMP method to test OXA-23 gene of CRAB was established in this research was simple,fast,sensitive and specific.Therefore,it is especially suitable wider use at the grass-roots unit,and it is of great significance for selecting reasonable choice of antibiotics by clinical doctor.
出处
《国际检验医学杂志》
CAS
2015年第4期513-515,共3页
International Journal of Laboratory Medicine
基金
四川省卫生厅科研资助项目(120347)
泸州市科技计划资助项目[2012-S-37(15/29)]
