一种新的等温扩增技术检测阪崎肠杆菌

A New Isothermal Amplification Assay for the Detection of Enterobacter sakazakii

目的：建立一种新型阪崎肠杆菌快速筛选检测方法——交叉引物恒温扩增结合免疫金标检测方法。方法：针对阪崎肠杆菌16S-23SrDNA间区序列设计特异性引物及探针，建立交叉引物等温扩增法，利用免疫金标试纸条对结果进行检测。用18株阪崎肠杆菌及其他36株近源菌进行特异性试验；通过定量DNA、纯菌液计数、添加干扰菌检测进行灵敏度验证。结果：建立方法具有很好的特异性；增菌液检测灵敏度为10^1CFU／mL，DNA检测灵敏度为10^0fg／test。结论：建立的交叉引物恒温扩增结合免疫金标检测方法特异性好、灵敏度高，不需要复杂仪器，对口岸快速筛查、基层或偏远经济不发达地区顺利开展检测具有实际意义。

Objective： To establish a new rapid screening method for the detection of E. sakazakii by using cross-primer amplification combined with immuno-blotting analysis. Methods： On the basis of sequence analysis of 16S-23S rDNA internal transcribed spacer in E. sakazakii, specific primers and probes were designed and developed for cross-primer isothermal amplification and an immune colloidal gold test strip was used to test the amplification products. A total of 18 E. sakazakii strains and 36 relative strains were used to analyze the specificity of the developed method. In addition, its sensitivity was also validated by DNA quantification and enumeration of pure and mixed cell suspensions. Results： The developed method was highly specific and sensitive. The limit of detection was l0~ fg/test for genomic DNA and 101 CFU/mL for pure bacterial culture. Conclusion： This method is characterized by high sensitivity, specificity, easy operation, and no requirements for complex instruments. Therefore, it is very useful for rapid and screening and detection ofE. sakazakii.