巴尔通体MALDI-TOF MS数据库的建立与鉴定方法的评价

目的建立巴尔通体的基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)数据库和质谱鉴定方法,利用野生菌株进行验证、评价。方法应用MALDI-TOF MS采集巴尔通体ATCC标准菌株和国内流行菌株的质谱数据,每株菌采集24张的质谱图集,获得特定的蛋白指纹图谱,汇总成不同种巴尔通体的标准质谱图,建立巴尔通体MALDI-TOF MS标准数据库。比较乙醇-甲酸提取法和直接涂抹法对质谱鉴定结果的影响,并进行重复性实验评估方法的稳定性。进一步应用173株野生巴尔通体菌株评估该标准数据库和相应的质谱鉴定方法。结果应用汉赛巴尔通体、五日热巴尔通体和杆菌样巴尔通体等21种/亚种巴尔通体,共计200株多地区来源的地理菌株,建立了巴尔通体MALDI-TOF MS蛋白指纹图谱标准数据库。2种样品制备方法均可以达到正确鉴定标准,提取法质谱得分略高于直接涂抹法。应用8种、16株巴尔通体菌株进行的重复性验证,得分为(9.078±0.031)~(9.776±0.006)分,变异系数(CV)为0.06%~1.12%。14种、173株巴尔通体测试菌株的质谱鉴定得分为9.014~9.796分,平均(9.462±0.195)分,在种水平上能被正确识别。结论基于该研究自主建立的巴尔通体质谱数据库,MALDI-TOF MS技术能够快速、准确鉴定巴尔通体种类,该课题组所建立的巴尔通体质谱标准数据库具有重要应用价值,对临床上巴尔通体病的实验室检验与诊断具有重要意义。

Identification of Bartonella species by MALDI-TOF mass spectrometry

Objective To establish a matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS)database and mass spectrometry identification method for Bartonella species which were verified and evaluated by using the wild strains of Bartonella spp.Methods We explored MALDI-TOF MS method to collect the data of the ATCC standard and domestic epidemic strains of Bartonella spp.,which comprising specific protein fingerprints for each strain from 24 consensus spectrum obtained for each species.The database of MALDI-TOF MS for Bartonella spp.was created using the standard mass spectra of different Bartonella species.We compared the ethanol-formic acid extraction and the direct transfer methods for sample preparation and performed repeatability experiments to evaluate the stability of the method.Furthermore,173 wild Bartonella strains were used to evaluate the MS database and the corresponding mass spectrometric method for Bartonella identification.Results A standard protein fingerprints database of MALDI-TOF MS for Bartonellae was established by using a total of 200 strains from multiple geographical origins,representing 21 recognized species/subspecies of Bartonella,including B.henselae,B.quintana,B.bacilliformis and the others.Both of the two methods of sample preparation were able to be employed to correctly identify the strains,and the MS scores from the ethanol-formic acid extraction method were slightly higher than those from the direct transfer method.The average scores of MS ranged from(9.078±0.031)to(9.776±0.006)and the CV values were between 0.06%and 1.12%from the data of reproducibility in intra-and inter-run experiments using 16 strains representing 8 species of Bartonella.One hundred and seventy-three tested Bartonella strains,representing 14 Bartonella species were accurately identified at the species level with identification scores ranging from 9.014 to 9.976(9.462±0.195).Conclusion MALDI-TOF MS technology can be used to identify Bartonella strains at the species level quickly and accurately based on an independently established database of mass spectra for Bartonella spp.in this study.Our standard database of Bartonella mass spectrometry plays an important role in testing and diagnosis of Bartonella disease in clinical microbiology laboratory.