基于MALDI-TOF MS技术快速鉴定奶牛乳腺炎奶样中疑似金黄色葡萄球菌

Rapid identification of suspected Staphylococcus aureus in cow mastitis milk based on MALDI-TOF MS technology

基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术对呼和浩特地区13家牧场257份奶牛乳腺炎奶样中分离的45株疑似金黄色葡萄球菌(Staphylococcus aureus,S.aureus)进行快速鉴定及同源性分析。采用甲酸-乙腈提取法对分离45株疑似菌株进行样品制备。通过MALDI-TOF MS获取特异性蛋白质图谱,并结合分析软件对获取的图谱进行鉴定及聚类分析;同时选用荧光定量PCR和nuc基因PCR扩增及序列分析方法进行辅助鉴定。结果显示,MALDI-TOF MS、荧光定量PCR和nuc基因PCR扩增及序列分析3种鉴定方法一致性将其中43株分离株鉴定为S.aureus,准确率100%(43/43),MALDI-TOF MS将另2株疑似菌株鉴定为产色葡萄球菌。聚类分型结果表明,在差异程度500时,MALDI-TOF MS可将鉴定的43株S.aureus分为5型,且各型内菌株大多数来源于各自牧场中,说明同一牧场内多数菌株分子水平差异较小,存在一定程度的同源关系。结果表明,MALDI-TOF MS具有快速、准确、高通量等优点,可以实现对S.aureus快速鉴定和分型,并通过对污染源进行溯源分析,为预防和控制S.aureus引发奶牛乳腺炎提供技术支持。

Based on matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS),45strains of suspected Staphylococcus aureus isolated from 257cow mastitis milk samples from 13pastures in Hohhot area were rapidly identified and their homology was analyzed.Sample preparation of 45suspected strains were isolated by formic acid acetonitrile extraction method.The specific protein maps were obtained through MALDI-TOF MS,and the obtained maps were identified and cluster analyzed with analysis software.At the same time,fluorescence quantitative PCR and nuc gene PCR amplification and sequence analysis were used for auxiliary identification.The results showed that the three identification methods of MALDI-TOF MS,fluorescent quantitative PCR and nuc gene PCR amplification and sequence analysis were consistent.Forty-three isolates were identified as Staphylococcus aureus,with an accuracy of 100%(43/43).MALDI-TOFMS identified the other 2suspected strains as Staphylococcus chromogenes.The cluster typing results showed that when the degree of difference was 500,MALDI-TOFMS could classify 43strains of Staphylococcus aureus identified into 5types,and most of the strains in each type came from their own farms,indicating that most strains in the same farm had little difference at the molecular level,and there was a certain degree of isogenic relationship.MALDI-TOF MS has the advantages of fast,accurate and high-throughput,which can realize the rapid identification and typing of Staphylococcus aureus,and provide technical support for the prevention and control of cow mastitis caused by Staphylococcus aureus through the traceability analysis of the pollution source.