基质辅助激光解吸电离飞行时间质谱在幽门螺杆菌鉴定中的应用

摘要

目的：优化幽门螺杆菌（ H.pylori）基质辅助激光解吸电离飞行时间质谱（ MALDI-TOF MS）检测前处理方法，建立MALDI-TOF MS的H.pylori 超级图谱并验证其鉴定能力。方法收集2015年1月至2016年7月期间复旦大学附属华东医院临床胃活检组织样本469例，进行H.pylori分离培养和鉴定，并利用16 S rRNA测序验证，共获得91株H.pylori菌株用于后续MALDI-TOF MS检测。在进行MALDI-TOF MS检测前，比较50％异丙醇、甲酸乙腈（1∶1）以及70％甲酸用于H.pylori前处理的效果。从91株临床分离的H.pylori菌株中随机选取40株进行MALDI-TOF MS检测，再进一步分别随机抽取原始图谱分为5株组、10株组、20株组和30株组，每组重复3次，采用SARAMIS Premium软件分析，建立超级图谱。剩余的51株H.pylori菌株用于验证新建超级图谱的鉴定能力。结果70％甲酸进行前处理效果最好。30株组建立的超级图谱的鉴定率最高，可达90.2％。用于验证的306张质谱图中46.1％的图谱取得了高可信度（≥90％）的鉴定结果、22.2％的图谱取得了中低度可信的鉴定结果，31.7％的图谱无鉴定结果。结论本研究优化了MALDI-TOF MS鉴定H.pylori的前处理方法，并建立了H.pylori超级图谱，提高了MALDI-TOF MS对于H.pylori的鉴定能力，为临床上快速准确地鉴定H.pylori提供了新的检测平台。（中华检验医学杂志，2017，40：31-35）%Objective To optimize the pre-treatment method before detecting Helicobacter pylori ( H.pylori ) by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry ( MALDI-TOF MS) and evaluate the ability of the Superspectra by MALDI-TOF MS for identifying clinical isolated H.pylori strains.Methods H.pylori were isolated from 469 biopsy samples of gastric mucosa collected from January 2015 to July 2016 in Huadong Hospital affiliated to Fudan University , 16 s rRNA sequencing were then performed to validate the strains.Then 91 isolated H.pylori strains were used for the subsequent MALDI-TOF analysis.The effect of pre-treatment of 50%isopropanol, formic acid and acetonitrile (1∶1), 70%formic acid were compared before H.pylori detecting by MALDI-TOF MS.40 out of 91 clinical H.pylori strains were detected by MALDI-TOF MS and the spectra were randomly assigned to 4 groups including 5, 10, 20, 30 spectra, each group had 3 repeats.Then 4 groups with different amount of spectra were used for creating Superspectra with SARAMIS Premium software , respectively.The remaining 51 H.pylori strains including 306 spectra were used for validating the identification rate of the Superspectra.Results With the use of 70%formic acid, the greater number of ion signals and higher relative intensity of the main peaks were observed than other pre-treatment reagents.The identification rate of Superspectra created by 30 strains group was the highest ( 90.2%).Among the 306 spectra, 46.1% of them achieved highly reliable identification , 22.2% achieved lower degree of reliable identification , and 31.7% of them achieved “no identification”.Conclusions The study optimized the pre-treatment method before H.pylori detecting by MALDI-TOF MS.The Superspectra was created with the good ability to rapidly identify clinical isolated H.pylori strains.