聚合酶链式反应和基质辅助激光解吸电离飞行时间质谱方法鉴定克罗诺杆菌

Application of polymerase chain reaction and matrix-assisted laser desorption/ionization time of flight mass spectrometry methods in Cronobacter rapid identification

目的采用聚合酶链式反应(PCR)和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术快速鉴定克罗诺杆菌的方法,实现对该菌显色培养基上生长的可疑菌落高通量、低成本初筛。方法通过内转录间隔区(its)基因设计针对克罗诺杆菌的属特异性引物,以7株参考菌株为对照,对2013年国家食品安全污染物监测网上报、初步鉴定为克罗诺杆菌的214株菌株进行复核鉴定。同时随机挑选其中84株菌,采用MALDI-TOF-MS技术和VITEK革兰阴性细菌鉴定卡鉴定。结果 7株参考菌株的PCR结果与对应大小一致,214株待测菌株PCR结果均为阳性。84株菌MALDI-TOF-MS和VITEK鉴定的结果均为克罗诺杆菌,符合率为100%。结论 PCR和MALDI-TOF-MS技术具有高通量、低成本、耗时短等优势,可以实现大量可疑菌落的初筛,为我国克罗诺杆菌的监测和防控提供技术支持。

Objective To develop two rapid identification method of Cronobacter use polymerase chain reaction(PCR) and matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS), and to achieve high-throughput and low-cost screening of suspicious colonies of Cronobacter on chromogenic medium. Methods A pair of genus specific primers for Cronobacter were designed by internal transcribed spacer(its) gene, and 7 standard strains were set as reference strains to re-identify 214 strains which were preliminarily identified as Cronobacter collected in 2013. 84 isolates were selected randomly and identified by MALDI-TOF-MS and VITEK gram-negative bacteria identification card. Results The molecular weight of 7 reference strains were the same as expected, and PCR result of 214 isolates were positive. All 84 strains were identified as Cronobacter by both MALDI-TOF-MS and VITEK, and the coincidence rate was 100%. Conclusion PCR and MALDI-TOF-MS have the advantages of high-throughput, low-cost and short time consuming, which could achieve the preliminary screening of a large number of suspicious colonies, and could provide technical support for Cronobacter surveillance and control in China.