基于荧光毛细管电泳的全序列多重PCR联合无细胞蛋白表达系统检测细菌碳青霉烯酶的研究

Carbapenem-resistant bacteria have now been reported to be challenging for clinical therapy. The variety and mutable carbapenemases play the most important role on drug-resistance. The current phenotypic and molecular identification methods are time-consuming and labor-intensive. What’s more, they are not suitable for identifying new gene subtypes and analyzing the activities of carbapenemases. Therefore, it is necessary to develop a rapid, cost-effective, sensitive, and high throughput method to investigate the distribution of carbapenemases genes in clinical specimens. In this project, we are aimed to develop a laser-induced fluorescence capillary electrophoresis analyzer based whole sequence multiplex PCR assay to rapidly identify the popular 46 kinds of carbapenemases genes including the mutations. Then, a cell-free in vitro protein expression system is going to be developed to further analyze the activities of carbapenemases coded by positive gene and new subtypes. Then, the sensitivity of the multiplex assay is going to be analyzed with recombinant plasmids containing the target genes. The specificity of the system is going to be evaluated with the target genes and some interference genes. Finally, the combined system is used to detect carbapenem-resistant bacteria in clinical specimens, and to assessment the clinical utility. This combined detection system will be rapidly and accurately determine the distribution of genes and the hydrolytic activity of carbapenemases. This method may be adopted for general use for molecular epidemiologic survey of resistance genes and for researching the drug-resistance mechanism. The results may offer a directory suggestion for clinical antibiotic therapy.

碳青霉烯类抗生素作为临床抗感染治疗的最后一线药物，该类抗生素耐药给临床治疗带来极大困难，碳青霉烯酶的产生是细菌对该类抗生素耐药的最主要机制。而碳青霉烯酶种类繁多且各种酶的基因亚型多变，现有的表型鉴定方法和分子生物学鉴定方法在分析时效、通量及新基因亚型检测方面存在较大的缺陷。本项目拟基于激光诱导荧光毛细管电泳分析仪建立一种由荧光标记通用引物和特异性嵌合引物介导的全序列荧光多重PCR体系，用于快速检测目前流行的46种碳青霉烯酶基因。针对多重检测体系筛选到的阳性基因及突变亚型，建立无细胞蛋白表达体系，体外合成碳青霉烯酶并验证其对亚胺培南的水解活性。然后，进行灵敏度和特异性分析；用于临床标本检测，评估其诊断效能。这种联合检测系统将快速、准确地确定临床感染性标本中碳青霉烯酶基因的分布以及其酶的水解活性，为耐药基因的分子流行病学研究及耐药机制研究提供新手段，为临床治疗采取行之有效的抗感染措施提供参考。

碳青霉烯酶的产生是细菌对碳青霉烯类抗生素耐药的最主要机制。碳青霉烯酶种类繁多，各种酶的基因亚型多变，且新的基因亚型不断涌现。现有的表型鉴定方法和分子生物学方法在分析时效、通量及新基因亚型检测及酶活性分析方面存在一定的缺陷。针对上述问题，本项目建立了三种不同的检测系统，具体包括：1、建立了三组全序列荧光定量PCR检测体系，快速检测17种碳青霉烯酶基因各个亚型，并通过阳性克隆质粒和临床样本，证实该检测体系适用于临床原始标本检测，可比传统方法提前48小时为临床提供抗生素耐药信息。2、针对临床广泛流行的6种碳青霉烯酶基因，建立了基于激光诱导毛细管电泳分析仪的多重耐药基因检测体系，其检测下限达到10 copies/反应。通过单管多重高通量检测，大大节约试剂和样本，减少工作量，满足大样本临床筛查和分子流行病学调查的要求。3、采用无细胞蛋白表达系统体外合成三种碳青霉烯酶并验证其水解活性，为新的碳青霉烯酶基因亚型的检测和活性分析提供了研究方法，同时也为特定蛋白质的合成和生物功能学研究提供的参考思路，在抗体和疫苗研发方面可具有一定实用价值。本项目的实施为耐药菌的早期诊断提供新方法，为临床合理有效使用抗生素提供重要依据。