探究乳粉中阪崎肠杆菌活菌检测方法灵敏度和检出限

Study on sensitivity and detection limit of detection method for Enterobacter sakazakii live bacteria in milk powder

中文关键词:  阪崎肠杆菌  活菌检测  转录-聚合酶链反应

英文关键词:Enterobacter sakazakii  live bacteria detection  reverse transcription-polymerase chain reaction

中文摘要:

      目的 探究乳粉中阪崎肠杆菌活菌检测的灵敏度和最低检出线的实时荧光逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)方法。方法 将羊奶粉作为基质, 根据GB 4789.40-2016中第一法中规定的增菌方法, 一组加入10CFU阪崎肠杆菌, 另一组加入等量的阪崎肠杆菌、大肠杆菌、鼠伤寒沙门氏菌、普通变形杆菌、粪肠球菌, 培养时间分别设置为15 h和18 h, 将不同培养时间的培养物分别取1 mL进行RNA提取, 以cDNA为模板进行扩增。结果 增菌培养18 h后, 提取RNA采用实时荧光RT-PCR技术, 结果显示扩增曲线良好, Ct值稳定。结论 乳粉中阪崎肠杆菌活菌检测, 增菌培养18 h后, 采用实时荧光RT-PCR, 最低可检出10 CFU的阪崎肠杆菌。

英文摘要:

      Objective To explore the sensitivity and the lowest detection line of Enterobacter sakazakii in milk powder by the real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR) method. Methods Goat milk powder was used as matrix. According to the enrichment method specified in the first method in GB 4789.40-2016, one group was added with 10 CFU of Enterobacter sakazakii, and the other group was added with the same amount of Enterobacter sakazakii, Escherichia coli, Salmonella typhimurium, Proteus vulgaris and Enterococcus faecalis. The culture time was set at 15 h and 18 h respectively. The cultures with different culture time were respectively taken 1 mL for RNA extraction and cDNA was used as template for amplification. Results After 18 h of enrichment culture, RNA was extracted by real-time fluorescence RT-PCR. The results showed that the amplification curve was good and the Ct value was stable. Conclusion After 18 h of enrichment culture, real-time fluorescence RT-PCR can detect at least 10 CFU of Enterobacter sakazakii viable bacteria in milk powder.