[关键词]
[摘要]
为实现奶粉中快速检测阪崎肠杆菌,本文建立了检测阪崎肠杆菌的恒温实时荧光法。针对阪崎肠杆菌16S rRNA设计三组LAMP引物,采用Deaou-308C恒温实时荧光检测平台,选取常见病原菌标准株进行引物特异性检测;选取阪崎肠杆菌标准菌株进行基因组DNA灵敏度和最低检测限测定,同时利用人工污染方式检测此方法在脱脂和全脂奶粉中的灵敏度和最低检测限,利用RealAmp法和国标法对20份市售奶粉进行对比实验。结果显示,引物组16S-11扩增效率最优,与常见病原菌无交叉反应,对阪崎肠杆菌基因组DNA、阪崎肠杆菌污染的脱脂和全脂奶粉的灵敏度分别达到102 CFU/mL、102 CFU/mL和103 CFU/mL;对阪崎肠杆菌基因组DNA和阪崎肠杆菌污染的脱脂和全脂奶粉的最低检测限分别达到103 CFU/mL、103 CFU/mL和104 CFU/mL;在20份市售奶粉样品中RealAmp检测结果与传统国标培养结果一致,表明本文建立的阪崎肠杆菌RealAmp检测方法适用于阪崎肠杆菌的快速检测。
[Key word]
[Abstract]
In order to rapidly detect Enterobacter sakazakii in milk powder, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) method was developed. Three sets of loop-mediated isothermal amplification (LAMP) primers targeting the Enterobacter sakazakii 16S rRNA sequence were designed. With a DEAOU-308C thermostat fluorescence detector, the specificity of the primers was examined using a standard strain of Enterobacter sakazakii. The sensitivity and detection limit on genomic DNA were also determined with a standard strain of Enterobacter sakazakii, followed by measuring the sensitivity and detection limit of this assay for artificially contaminated skimmed milk powder and whole milk powder, and a comparative experiment was performed on 20 commercial milk powder samples using the RealAmp method and national standard method. The results showed that the primer set 16S-11 had the best amplification efficiency, showed no cross-reactivity with common pathogens, and the sensitivity of RealAmp for detecting Enterobacter sakazakii genomic DNA, artificially contaminated skimmed milk powder, and whole milk powder reached 102 CFU/mL, 102 CFU/mL, and 103 CFU/mL, respectively. The detection limit of RealAmp in detecting Enterobacter sakazakii genomic DNA, artificially contaminated skimmed milk powder, and whole milk powder reached 103 CFU/mL, 103 CFU/mL ,and 104 CFU/mL, respectively. The results of 20 commercial milk powder samples obtained by the RealAmp method were consistent with those from the national standard culture method. The results suggest that this RealAmp assay is suitable for the rapid detection of Enterobacter sakazakii in milk powder.
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[基金项目]
中央高校基本科研业务费专项资金资助(2015ZM063);中国博士后科学基金(2015M580723)