Background In recent years, under the selective challenge of antibiotics, the variety and number of drugresistant pathogenic microorganisms have increased significantly, which brings great challenges to clinical diagnosis and treatment, especially the infection caused by carbapenem resistant Enterobacteriaceae (CRE). Carbapenemases production is the main mechanism of drug resistance of Enterobacteriaceae to carbapenems. Drug resistance of Carbapenems can be caused by three mechanisms, resulting in the production of five major Carbapenemases. These are Klebsiella pneumoniae enzyme (KPC), New Delhi metal β-lactamase (NDM), carbapenem hydrolyzed oxalase (OXA-48 like), integrin-encoded metal β- lactamase (VIM) and IMP (Imipenemase). The real-time fluorescence quantitative PCR (qPCR) method based on molecular beacons was combined with the melting curve analysis to identify five drug resistance genes simultaneously by a single PCR reaction, with rapid detection, high sensitivity and strong specificity. Based on this principle, we developed the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae (Dynamiker Biotechnology (Tianjin) Co., Ltd.). Methods We evaluated the performance of the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae, including the limit of detection (LoD) and cross-reactivity, and compared it with the lateral flow immunochromatography assay (LFA). Results The LoD ranged from 75-450 CFU/mL for the five carbapenemase genes. The analytical specificity for target genes was 100%, as assessed with a panel of 15 pathogens, which indicated no cross-reactions. Comparison of qPCR and LFA results from twenty-three CRE clinical isolates with characterized carbapenemase content demonstrated a complete agreement (Table 1). Conclusions The novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae is an accurate and rapid method to identify KPC, NDM, VIM, IMP and OXA-48-like carbapenemases in the clinical microbiology laboratory, which can guide infection control programs to limit the spread of these organisms.

中文翻译：

---

B-205 用于检测产碳青霉烯酶肠杆菌科细菌的新型荧光测定法的性能


背景近年来，在抗生素的选择性挑战下，耐药病原微生物的种类和数量显着增加，给临床诊断和治疗带来巨大挑战，特别是碳青霉烯类耐药肠杆菌（CRE）引起的感染。产生碳青霉烯酶是肠杆菌科细菌对碳青霉烯类药物产生耐药性的主要机制。碳青霉烯类药物的耐药性可由三种机制引起，从而产生五种主要的碳青霉烯类酶。它们是肺炎克雷伯菌酶（KPC）、新德里金属β-内酰胺酶（NDM）、碳青霉烯水解草酸酶（OXA-48 样）、整合素编码的金属β-内酰胺酶（VIM）和IMP（亚胺培南酶）。基于分子信标的实时荧光定量PCR（qPCR）方法与熔解曲线分析相结合，可通过一次PCR反应同时鉴定5个耐药基因，检测速度快、灵敏度高、特异性强。基于这一原理，我们开发了新型荧光检测方法，用于快速检测多重耐药肠杆菌科细菌中的碳青霉烯酶（德纳米克生物技术（天津）有限公司）。方法 我们评估了用于快速检测多重耐药肠杆菌科细菌中碳青霉烯酶的新型荧光测定法的性能，包括检测限（LoD）和交叉反应性，并将其与侧流免疫层析测定法（LFA）进行比较。结果 5 个碳青霉烯酶基因的 LoD 范围为 75-450 CFU/mL。根据一组 15 种病原体的评估，目标基因的分析特异性为 100%，表明没有交叉反应。 对具有碳青霉烯酶含量特征的 23 个 CRE 临床分离株的 qPCR 和 LFA 结果进行比较，结果显示完全一致（表 1）。结论 新型荧光法快速检测多重耐药肠杆菌科细菌中的碳青霉烯酶，是临床微生物实验室中准确快速鉴定 KPC、NDM、VIM、IMP 和 OXA-48 类碳青霉烯酶的方法，可指导感染控制项目限制这些生物体的传播。