质谱SARAMIS软件比对分析功能快速筛查高毒力肺炎克雷伯菌的效果评估  

Effectiveness evaluation for rapid screening of hypervirulent Klebsiella pneumoniae by comparison analysis of MS SARAMIS software

作　　者：王惠 温海楠[1] 张宗伟 闫妹姝[1] 张盼 梁悦怡 刘焱超 谢守军[1] WANG Hui;WEN Hainan;ZHANG Zongwei;YAN Meishu;ZHANG Pan;LIANG Yueyi;LIU Yanchao;XIE Shoujun(Department of Clinical Laboratory,Affiliated Hospital of Chengde Medical College,Chengde 067000,Hebei,China)

机构地区：[1]承德医学院附属医院南区检验科,河北承德067000

出　　处：《临床检验杂志》2022年第11期857-862,共6页Chinese Journal of Clinical Laboratory Science

基　　金：承德市科技计划项目(201804A082)。

摘　　要：目的评价基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)SARAMIS软件快速区分高毒力肺炎克雷伯菌(hvKP)与经典肺炎克雷伯菌(cKP)的能力。方法收集2020年11月—2021年10月承德医学院附属医院临床分离非重复肺炎克雷伯菌(KP)共199株,采用MALDI-TOF MS技术对KP菌株进行鉴定。PCR扩增peg-344、rmpA和iucA毒力基因,以上述3种基因同时检测阳性作为hvKP区别于cKP的诊断标准。随机取其中90株KP分为hvKP(47株)组和cKP(43株)组,建立快速鉴别hvKP和cKP的筛选模型。菌株经梅里埃质谱仪检测并采集蛋白质图谱,收集两组细菌的蛋白质图谱导入质谱科研库SARAMIS软件进行修饰放大比对分析,肉眼观察上述图谱筛选出有明显差异的蛋白质峰,分别计算有差异的质荷比处蛋白质峰面积(area,A)与强度(intensity,I)的比值(A/I值)。将两组细菌的A/I值导入SPSS 26.0软件进行统计分析,利用受试者工作特征(ROC)曲线评估上述有统计学差异A/I值的临床诊断效果。采取盲法对另外109株菌株进行验证研究,评价A/I研究指标鉴别hvKP和cKP的诊断价值。结果peg-344、rmpA、iucA毒力基因检测显示hvKP占全部菌株的39.7%。筛选模型中,hvKP和cKP两组细菌的相同质荷比处有明显A/I值差异的蛋白质峰分别位于质荷比4154±5、4185±5、4366±5、4765±5处,hvKP和cKP组间比较差异有统计学意义(P<0.01)。ROC曲线结果显示,质荷比4154处蛋白质峰的A/I值的曲线下面积(AUC)为0.892,A/I值的临界值为47.87时约登指数最大,诊断效能最高,其敏感性为91.5%,特异性为86.0%,其余3处质荷比蛋白质峰A/I值的AUC均<0.5,诊断价值不高。以质荷比4154处A/I值为研究指标,对另外109株菌进行盲测检验,结果显示质荷比4154处A/I值≥47.87时区分hvKP与cKP的敏感性和特异性分别为87.5%、85.7%。结论质谱技术能较快速、准确地区分hvKP和cKP,质荷比4154处A/I值≥47.87有望成为预测hvKP的潜在研究指�Objective To evaluate the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS)SARAMIS software for distinguishing hypervirulent Klebsiella pneumoniae(hvKP)from classical Klebsiella pneumoniae(cKP)rapidly.Methods A total of 199 strains of Klebsiella pneumoniae(KP)were collected in the Affiliated Hospital of Chengde Medical College from November 2020 to October 2021,and all the KP strains were identified by MALDI-TOF MS.The virulence genes,peg-344,rmpA and iucA were amplified by PCR.The diagnostic criterion of distinguishing hvKP from cKP was simultaneously positive for the above three genes:peg-344,rmpA and iucA.Ninety strains of KP were randomly divided into hvKP group(47 strains)and cKP group(43 strains),and a screening model was established for rapid identification for hvKP and cKP.The strains were tested by bioMerieux Vitek MS mass spectrometry system and their protein spectra were acquired.The spectral data of the two groups of bacteria were collected and imported into SARAMIS software of mass spectrometry research database for modification,amplification and comparison analysis.The protein peaks with obvious differences were screened by simple visual observation and the area/intensity(A/I)ratio of protein peak at different m/z ratios was calculated.The A/I values of the two groups of bacteria were imported into SPSS 26.0 software for statistical analysis.The receiver operating characteristic curve(ROC)was used to evaluate the clinical diagnostic efficiency of the above-mentioned statistically different A/I values.Another 109 strains were verified by blind method to evaluate the diagnostic efficacy of A/I values for distinguishing hvKP from cKP.Results The analysis for virulence gene peg-344,rmpA and iucA showed that hvKP accounted for 39.7%(79/199)of all the strains.In the screening model,the protein peaks of hvKP and cKP groups with obvious difference of A/I value in the same m/z ratio were found at 4154±5,4185±5,4366±5 and 4765±5,respectively and the diff

关 键 词：高毒力肺炎克雷菌 经典肺炎克雷伯菌 毒力基因 质荷比蛋白质峰峰面积/强度 质谱仪 SARAMIS软件 

分 类 号：R446.5[医药卫生—诊断学]