基于MALDI-TOF/TOF技术的单增李斯特菌四种血清型的快速鉴别

单增李斯特菌是一种重要的人畜共患病致病菌，其传统血清型鉴定方法具有耗时长、操作繁琐等缺点，现行的多重PCR方法无法精确判定其血清型。基于基质辅助激光解析/电离飞行时间质谱（MALDI-TOF/TOF）的快速、准确、高通量等特点，应用该技术建立了单增李斯特菌4种血清型的鉴别方法，以实现食品样品中单增李斯特菌4种血清型的高通量快速甄别。采用优化的MALDI-TOF/TOF条件对4种血清型的单增李斯特菌标准菌株和分离菌株进行检测，发现单增李斯特菌4种血清型的质谱特征峰。选取最优的支持向量机算法，建立基于1/2a、1/2b、1/2c和4b血清型差异的判别模型，运用该模型鉴别4种血清型的单增李斯特菌。初步建立了单增李斯特菌4种血清型的MALDI-TOF/TOF快速鉴别方法。以4种血清型的单增李斯特菌标准菌株为阳性对照、144株食品分离株为检测对象，运用该方法对单增李斯特菌1/2a、1/2b、1/2c、4b四种血清型鉴定符合率分别达到92.7%、90.3%、100%、100%，多重PCR方法的符合率为71.4%、87.0%、96.2%、95.0%。可见,建立的MALDI-TOF/TOF方法可对4种血清型的单增李斯特菌快速鉴别。与多重PCR方法相比，该方法具有快速、高通量、重现性好等特点，可用于食品中单增李斯特菌血清型的快速鉴定。

Rapid Identification of Four Serotypes of Listeria Monocytogenes Using MALDI-TOF/TOF Technique

Listeria monocytogenes is an important zoonotic pathogenic bacteria. The traditional serological identification method is time consuming and cumbersome to operate, and current multiplex polymerase chain reaction (PCR) methods cannot accurately determine its specific serotype. Based on the quick, accurate and high-flux, etc. characteristics of matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF), this paper established an identification method for 4 serotypes of L. monocytogenes, so as to distinguish the L. monocytogenes in food sample in a large quantity with high speed. The isolated and standard strains of 4 serotypes of L. monocytogenes were detected under the optimized MALDI-TOF/TOF condition, and the characterisic peaks of mass spectrum of 4 L. monocytogenes serotypes were discovered. This paper selected the optimal support vector machine (SVM) algorithm and established discriminant model of serotype difference based on 1/2a, 1/2b, 1/2c and 4b, and identified 4 L. monocytogenes serotypes by this model. Thus, a fast MALDI-TOF/TOF identification method for 4 L. monocytogenes serotypes was preliminarily established. Taking these 4 standard strains as positive control and 144 strains isolated from food for detect objects, this paper identified 4 L. monocytogenes serotypes by this method, and the accuracy rates of 1/2a, 1/2b, 1/2c and 4b reached 92.7%, 90.3%, 100% and 100%, respectively. The accuracy rate of multiplex PCR were 71.4%, 87.0%, 96.2% and 95.0%, respectively. Visibly, the established MALDI-TOF/TOF typing method could be used for rapid identification of 4 L. monocytogenes serotypes. Compared with multiplex PCR method, this method had the characteristics of quick, high-flux, good reproducibility, etc., so it could be used for fast serotype identification of L. monocytogenes in food.