摘要
目的建立以液态芯片为检测手段的快速、高通量的铜绿假单胞菌多重耐药基因监测技术平台,探索建立能够常规应用于临床并解决现行临床表型测定局限性的分子生物学技术。方法针对铜绿假单胞菌中常见的OPRD2、TEM、OXA10、AAC3、AAC6、VIM、VEB、IMP共8个耐药基因序列进行分析,依次对这8个耐药基因设计多重PCR扩增引物和序列特异的核酸探针,选取临床鉴定的含各耐药基因的耐药菌株作为阳性参比品进行检测工艺优化,进行铜绿假单胞菌多重耐药基因液态芯片监测平台的研发。结果初步建立了铜绿假单胞菌多重耐药基因液态芯片监测平台,组装了铜绿假单胞菌多重耐药检测液态芯片试剂盒1套。选择了8株含各耐药基因铜绿假单胞菌阳性菌株,用组装的试剂盒重复检测10次,耐药基因检测阳性率为100%,各耐药基因检测信号间无交叉,灵敏度﹑特异度以及重复性都很好。利用此平台进行了一定数量的临床标本检测分析。从检测结果来看,40份标本中,18份标本耐药基因阳性,其中含多耐药基因的标本有9份,占50%。18份阳性标本的耐药基因与耐药表型分析结果一致。结论此监测平台是将一种多重扩增技术和芯片技术-悬浮阵列技术相结合的方法,具有特异性高、可组合筛查、检测通量高的优势。
Objective To design a liquid chip for rapid, high-throughput detection of multidrug resistance genes in Pseudomonas aeruginosa. Methods Based on the sequence the common resistance genes of OPRD2,TEM,OXA10,AAC3,AAC6,VIM,VEB and IMP in Pseudomonas aeruginosa, the specific primer pairs and probes were designed. Using the clinical isolateswith single resistance gene of resistant strains as positive references, the suspension array detection platform of multidrug resistance genes in Pseudomonas aeruginosa was studied. Results The suspension array detection platform of multidrug resistance genes in Pseudomonas aeruginosa was initially established. At the same time, the liquid chip kit of detection multidrug resistance genes in Pseudomonas aeruginosa was prepared; the detection system was optimized and its specificity, sensitivity, repeatability were analyzed, which reached clinical application level.Conclusion This test platform is a method of multiple amplification technology combined with the most advanced and practical technology-suspension array technology, which has the unique advantages, such as high specificity, highthroughput and can be used for combined screening.
出处
《热带医学杂志》
CAS
2016年第6期732-736,共5页
Journal of Tropical Medicine
基金
青岛市民生科技计划资助项目[11-2-3-5(8)-nsh]
关键词
铜绿假单胞菌
耐药基因
液态芯片
Pseudomonas aeruginosa
Drug resistance gene
Liquid chip
