微生物饲料添加剂中丁酸梭菌的测定方法比较研究

A comparative study of different methodsto determine Clostridium butyricum in microbial feed additive

试验旨在建立快速准确测定微生物饲料添加剂中丁酸梭菌的方法,分别对不同计数培养基和菌株鉴定方法进行比较,并开展实际样品测定。结果显示,丁酸梭菌在亚硫酸铁琼脂、TSC琼脂和TSN琼脂中均可生长,但亚硫酸铁琼脂培养基的计数结果最高;微量生化鉴定管、16S rRNA扩增测序、荧光定量PCR及基质辅助激光解析电离质谱(MALDI-TOF MS)均能鉴定丁酸梭菌,但荧光定量PCR方法最快速、准确、适用范围广;2批次市售样品分别含丁酸梭菌9.1×10^(8)、1.2×10^(9)CFU/g,典型菌落荧光定量PCR鉴定结果阳性。研究表明,亚硫酸铁琼脂培养基平板计数法结合荧光定量PCR可对微生物饲料添加剂中的丁酸梭菌进行快速有效的测定。

To establish a rapid and accurate method for the determination of Clostridium butyricum in microbial feed additives,different counting media and identification methods were compared and the samples in market were determined.The results showed that Clostridium butyricum could grow in sulfite iron agar,TSC agar and TSN agar,while the colony counting result of ferric sulfite agar medium was the highest.Clostridium butyricum could be identified by microbiochemical identification tubes,16S rRNA gene amplifying and sequencing,qPCR amplification and MALDI-TOF MS,while the qPCR amplification was the most rapid and accurate as well as widely applied method.Two samples from market were determind with results of 9.1×108,1.2×109 CFU/g respectively.Typical colonies were identified by qPCR with positive results.The study indicates that Clostridium butyricum in microbial feed additives can be effectively determined by ferric sulfite agar plate counting along with biochemical and molecular biological identification.