论文部分内容阅读
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 establish a rapid and high-throughput platform for the monitoring of multi-drug resistant genes of Pseudomonas aeruginosa with liquid chip as a detection method and to establish a molecular biological technique that can be routinely applied in clinic and to solve the limitations of current clinical phenotypic assays. Methods A total of 8 resistance gene sequences of OPRD2, TEM, OXA10, AAC3, AAC6, VIM, VEB and IMP in Pseudomonas aeruginosa were analyzed. Multiplex PCR amplification primers and Sequence-specific nucleic acid probes were selected, and drug-resistant strains containing each drug resistance gene clinically identified were selected as positive reference materials for optimization of the detection process. R & D of the liquid chip monitoring platform of the multi-drug resistant gene of Pseudomonas aeruginosa was carried out. Results A monitoring platform of Pseudomonas aeruginosa multidrug resistance gene liquid chip was initially established and one kit of Pseudomonas aeruginosa multi-drug resistant liquid chip kit was assembled. Eight strains positive for Pseudomonas aeruginosa were selected and tested with the kit for 10 times. The positive rate of drug resistance gene was 100%. There was no crossover, sensitivity, specificity Degree and repeatability are good. Use this platform for a certain number of clinical specimens testing and analysis. From the test results, of the 40 samples, 18 samples were positive for drug resistance gene, of which 9 samples containing multi-drug resistance genes accounted for 50%. The resistance genes of 18 positive specimens were consistent with the results of drug resistance phenotype analysis. Conclusion This monitoring platform is a combination of multiplex amplification and chip technology - suspension array technology. It has the advantages of high specificity, combinatorial screening and high detection throughput.