霍乱弧菌基质辅助激光解吸飞行时间质谱数据库的建立与应用

Establishment and application of matrix-assisted laser desorption ionization-time of flight mass spectrometry database for the detection of Vibrio cholerae

目的建立基质辅助激光解吸电离飞行时间质谱法(matrix-assisted laser desorption ionization time-of-flight mass spectrometry,MALDI-TOF-MS)快速检测霍乱弧菌,并建立数据库。方法考察培养基、培养时间、培养温度以及样品前处理方法对图谱质量和鉴定结果的影响,对方法的稳定性、重复性进行研究;采集101株霍乱弧菌食品分离株MALDI-TOF-MS指纹图谱,并对图谱进行分析。其中标准菌株CICC23794及40株分离株作为建库菌株,其余60株用于建库后的测试。结果采用2%NaCl-TSA培养基,在37℃、24h条件下培养,可达到强峰值信号,谱图重现性良好,甲酸-乙腈提取法获得的质谱图特征峰多,信噪比高;建库后,60株霍乱弧菌均可达到高于2.0的水平,其中约有20株霍乱弧菌鉴定分值可达到2.4以上。结论本研究建立的霍乱弧菌MALDI-TOF-MS鉴定方法结果准确,可用于实际检测。

Objective To establish a rapid method for the detection and identification of Vibrio cholerae by matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS)and establish the database.Methods In order to verify the stability and repeatability,different mediums,culture times and sample handing methods were optimized.The 101 isolates of Vibrio cholerae was used as a reference to evaluate the credibility of MALDI-TOF-MS method and analysis on mass spectrometric profiles.The reference strain CICC23794 and 40 isolates were used as the library-building strains,and the other 60 strains were used for the test after the library-building.Results Taking 2%NaCl-Tryptic Soy Agar Base as the culture medium,the mass spectrum with strong signal and good repeatability was acquired after 37℃,24 h.The results showed that more characteristic peak of protein with smooth baseline,less noise and higher signal-to-noise ratio were obtained by ethanol/formic acid treatment method.The 60 isolates of Vibrio cholera could be identified with a score of 2.0,and 20 strains of Vibrio cholerae could be identified with a score of 2.4 or higher.Conclusion MALDI-TOF-MS is a fast and accurate method for the identification of Vibrio cholera,and can be used for routine detection.