基于数值鉴定法的肠杆菌科生化试剂盒的研制及效果评价

【背景】肠杆菌科细菌是食源性和临床感染的主要病原菌，对人类和动物健康有重要影响，以API 20E为“金标准”的数值鉴定法是其主要的鉴定方法之一，但现有的数值鉴定方法存在手工单个加样或/和准确率低、价格贵的问题。【目的】研制出半自动化、准确性高及价格低的肠杆菌科细菌数值鉴定生化试剂盒。【方法】在本团队前期建立的肠杆菌科数值鉴定系统理论模型与配套软件的基础上，设计并优化筛选出的24种生化基质微量化配方，在此基础上，研制半自动冻干鉴定条；以现行商品化的数值鉴定条API 20E、MALDI-TOF MS及16S rRNA基因测序为对照，对研制的集半自动生化鉴定条与在线分析软件于一体的生化试剂盒进行效果评价。【结果】共获得458株肠杆菌科细菌的生化谱，在属水平总体鉴定准确率达到98.5%，在种水平总体鉴定准确率达到96.5%；仅需两次加样即可获得鉴定结果，且价格仅为API 20E的4.46%；产品保质期为7 d，生化试验具有稳定可重复性。【结论】本研究研制出的半自动化肠杆菌科细菌数值鉴定生化试剂盒简便经济、准确率高，为肠杆菌科细菌的鉴定提供技术支撑，并为其他科属细菌的数值鉴定生化产品的开发提供技术指引。

Development and performance evaluation of a biochemical kit for identification of Enterobacteriaceae based on numerical identification method

[Background] Bacteria of Enterobacteriaceae are the main pathogens of foodborne and clinical infections, posing a threat to human and animal health. The numerical identification method using API 20E as the “gold standard” is one of the main identification methods for Enterobacteriaceae. However, the existing numerical identification methods have problems such as laborious sample addition, low accuracy, and high prices. [Objective] To develop a semi-automatic, high-precision, and low-cost biochemical kit for identifying the bacteria of Enterobacteriaceae based on the numerical identification method. [Methods] On the basis of the theoretical model and supporting software of the numerical identification system for Enterobacteriaceae established by our team, we designed and optimized 24 biochemical matrix trace formulations. Furthermore, a semi-automatic freeze-drying identification strip was developed. Using the commercialized numerical identification strip API 20E, MALDI-TOF MS, and 16S rRNA gene sequencing as the controls, we evaluated the performance of the developed biochemical kit that integrated semi-automatic biochemical identification strips and online analysis software. [Results] The biochemical spectra of 458 strains of Enterobacteriaceae were obtained, with the overall identification accuracy of 98.5% at the genus level and 96.5% at the species level. The kit only needed twice sample addition to obtain the identification results, with the price only 4.46% that of API 20E. The shelf life of the product was 7 days, and the biochemical experiments had repeatability. [Conclusion] We develop a semi-automatic biochemical kit for identification of Enterobacteriaceae based on the numerical identification method, which has simple operation, a low cost, and high accuracy. This study provides technical support for the identification of Enterobacteriaceae and the development of biochemical products for numerical identification of other families and genera of bacteria.