基质辅助激光解吸电离飞行时间质谱技术对分枝杆菌液体培养系统菌种鉴定的临床研究

目的 应用基质辅助激光解吸电离飞行时间质谱(matrix assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF MS)对Bactec^TM MGIT^TM 960液体培养出的阳性培养物进行高通量、快速、准确的菌种鉴定,为临床早期、精准治疗提供病原学依据。方法 选取我院Bactec^TM MGIT^TM 960分枝杆菌快速液体培养的阳性培养物为研究对象,通过不同的孵育时间、不同大小的研磨珠处理、不同的破壁方式摸索出MALDI-TOF MS对Bactec^TM MGIT^TM 960阳性培养物鉴定的“最优处理方法”,并将所有结果与基因芯片法菌种鉴定做参照进行比对,鉴定不一致的结果通过基因测序确认。结果 Bactec^TM MGIT^TM 960液体培养报阳性后增加孵育时间可提高检出率及鉴定分值,同时使用0.5 mm的氧化锆珠研磨,使用超声震荡混匀处理,鉴定效果最佳。鉴定结果方面,液体系统报阳的420份阳性标本,基因芯片法鉴定结果为结核分枝杆菌占69.05%(290/420),非结核菌占28.81% (121/420),9例未鉴定出,未鉴定占2.14%(9/420),错误鉴定占0.95%(4/420),总鉴定率为97.86%(411/420);采用MALDI-TOF MS质谱鉴定420份阳性标本中鉴定出419例,其中结核分枝杆菌占69.05%(290/420),非结核占30.00%(126/420),星型奴卡菌0.48%(2/420),巴西奴卡菌占0.24%(1/420),1例未鉴定出,总鉴定率为99.76%。质谱鉴定分值≥2.0的400例占95.24%,分值在1.7~2.0的19例占4.52%,分值<1.7的1例占0.24%。结论 本实验实现了Bactec^TM MGIT^TM 960液体培养阳性培养物的直接质谱鉴定,缩短了培养鉴定的时间,将分枝杆菌鉴定到种水平,能区分出结核菌与非结核菌,同时也能将与分枝杆菌相似的奴卡菌鉴定出来,对临床疾病的鉴别诊断起到了积极的作用。

Clinical study on the identification of Mycobacterium liquid culture system by matrix assisted laser desorption ionization time of flight mass spectrometry

To identify the high-throughput, rapid and accurate strains of bactectm mgittm 960 by matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS), so as to provide etiological basis for early and accurate clinical treatment. The sputum and alveolar lavage fluid samples from tuberculosis and respiratory departments of our hospital were selected as the research objects. Through different incubation time, different size of abrasive beads and different ways of breaking the wall, we found out the effect of MALDI-TOF MS on bactectm mgittm 960 positive culture identification of the "optimal treatment method", and all the results were compared with the microarray method for species identification, identification inconsistent results were confirmed by gene sequencing. When bactectm mgittm 960 liquid culture was positive, increasing incubation time could improve the detection rate and identification score. At the same time, 0.5mm zirconia beads were used to grind and ultrasonic vibration was used to mix. The identification effect was the best.Among 420 were positive, of which 69.05% (290/420) were Mycobacterium tuberculosis, 28.81% (121/420) were non tuberculosis, 2.14% (9/420) were not identified, 0.95% (4/420) were false identified, and the total identification rate was 97.86% (411/420). 419 of 420 positive samples were identified by MALDI-TOF MS. Among them, M. tuberculosis accounted for 69.05% (290/420), non tuberculosis accounted for 30.00% (126/420), Nocardia stellaris accounted for 0.48% (2/420), Nocardia brasiliensis accounted for 0.24% (1/420), one case was not identified, the total identification rate was 99.2%. 95.23% of the 400 cases had a Appraisal score of > 2.0, 4.52% of the 19 cases had a score of 1.7-2.0, and 0.24% of the cases had a score of < 1.7. This experiment realizes the identification of baccetm mgittm 960 liquid culture positive culture by direct mass spectrometry, shortens the time of culture identification, identifies mycobacteria to species level, distinguishes tubercle bacillus from non tubercle bacillus, and identifies Nocardia similar to Mycobacteria, which plays a positive role in the differential diagnosis of clinical diseases. Mass spectrometry, a high-throughput means of microbial identification, plays a great role in promoting laboratory work, and its wide application can help the rapid diagnosis of tuberculosis Mass spectrometry, a high-throughput means of microbial identification, plays a great role in promoting laboratory work, and its wide application can help the rapid diagnosis of tuberculosis.