基于基质辅助激光解吸电离飞行时间质谱的 溶藻弧菌鉴定研究

Identification of Vibrio alginolyticus using matrix-assisted laser desorption ionization-time of flight mass spectrometry

中文关键词:  基质辅助激光解吸电离飞行时间质谱  SARAMIS分析软件  溶藻弧菌  鉴定

英文关键词:matrix-assisted laser desorption/ionization time-of-flight mass spectrometry  SARAMIS analysis software  Vibrio alginolyticus  identification

中文摘要:

      目的 建立快速检测和鉴定溶藻弧菌的方法, 采用基质辅助激光解吸电离飞行时间质谱(MALDI- TOF-MS)和SARAMIS分析软件, 对来自水产品中的溶藻弧菌进行了检测和鉴定。方法 将从样品中分离得到的可疑菌落于37 ℃纯化培养24、48、72 h后, 分别用微生物API生化鉴定系统和MALDI-TOF-MS方法进行分析鉴定, 并对MALDI-TOF-MS鉴定方法的稳定性、准确性和重复性进行初步探讨。结果 不同的培养时间, 24、48和72 h对MALDI-TOF-MS检测的蛋白质谱图影响较小, 鉴定值分别为94.10%, 93.90%, 92.70%; 同时用微生物API-20E生化鉴定试剂盒进行辅助分析鉴定, 培养24 h后, 鉴定值为97.7%, T值为0.47, 但培养72 h后, 鉴定值为52.4%, T值为0.15。同一样品点样2次, 得到的6张图谱显示出峰位置基本一致。用标准菌株大肠埃希菌ATCC 8739和溶藻弧菌ATCC 17749纯化培养24 h后, 鉴定值分别为99.90%和97.60%。结论 与传统的生化鉴定方法相比, MALDI-TOF-MS和SARAMIS软件分析方法具有较高的稳定性、重复性和准确性, 可以快速、准确地鉴定溶藻弧菌, 可用于水产品中溶藻弧菌的高通量、低成本的快速检测鉴定。

英文摘要:

      Objective To establish a method for the rapid detection and identification of Vibrio alginolyticus, and to detect and identify Vibrio alginolyticus carried in the aquatic product by matrix-assisted laser de-sorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and the SARAMIS analysis software. Methods Suspicious colony isolated from the samples were analyzed by API-20E identification system and MALDI-TOF-MS method after being purified and cultured for 24, 48, 72 h at 37 ℃. The accuracy, repeatability, and stability of MALDI-TOF-MS were also studied. Results Different time intervals of 24, 48 or 72 h were tested with mass spectrometry and little infects were found, with the identification scores of 94.10%, 93.90%, and 92.70%, respectively. Bacteria API-20E identification system was used to assist the analysis and identification. The results showed that the identification scores were 97.7% and 52.4%, and T value were 0.47 and 0.15, with the different time intervals of 24 and 72 h. The six mass spectrums of the same sample showed the peak positions were basically identical. The identification scores of standard strains of E. coli ATCC 8739 and V. alginolyticus ATCC 17749 after 24 h were 99.90% and 97.60 %, respectively. Conclusion Compared with the traditional biochemical identification method, MALDI-TOF-MS is stable, repeatable and accurate. Moreover, it is less time-consuming, and the cost for reagents is minimized. It can be used as a tool for rapid detection and identification of Vibrio alginolyticus carried in the aquatic product.