实时荧光环介导等温扩增技术检测婴儿配方奶粉中阪崎克罗诺杆菌的研究

Detection of Cronobacters akazakii in infant formula by real-time fluorescence loop-mediated isothermal amplification

中文关键词:  实时荧光  环介导等温扩增  阪崎克罗诺杆菌  婴儿配方奶粉

英文关键词:real-time fluorescence  loop-mediated isothermal amplification  Cronobacters akazakii  infant formula

中文摘要:

      目的 建立实时荧光环介导等温扩增法(real-time fluorescence loop-mediated isothermal amplification, RT-LAMP)检测阪崎克罗诺杆菌。方法 以阪崎克罗诺杆菌(基因号AY702093)16S~23S rRNA的保守序列进行引物设计, 对dNTPs、Mg2+及模板浓度等反应条件进行优化, 并考察该方法的特异性和灵敏度。结果 实时荧光LAMP法检测的最佳反应体系为: 内引物1.6 μL FIP和1.6 μL BIP, 外引物: 0.5 μL F3和0.5 μL B3, 2.5 μL2.5 mmol/L dNTP, 2 μL 4 mmol/L MgSO4, 3 μL Buffer, 1.6 μL 10×Bst DNA聚合酶, 3 μL DNA模板, 0.5 μL 100×荧光染料, 用灭菌双蒸水补足25 μL体系, 在63 ℃下反应60 min。除阪崎克罗诺杆菌之外其他菌株均没有产生特异性荧光扩增曲线。实时荧光LAMP检测克罗诺杆菌的灵敏度可达到8×10-2 CFU/mL。结论 本方法检测克罗诺杆菌具有耗时短、特异性强及灵敏度高等优点, 可为快速检测婴儿配方奶粉中的克罗诺杆菌提供参考。

英文摘要:

      Objective To establish a method for the determination of Cronobacters akazakii by real-time fluorescence loop-mediated isothermal amplification (RT-LAMP). Methods Using 16S~23S rRNA conserved sequence of Cronobacter sakazakii as target sequences for primer design, the reaction conditions including dNTPs, Mg2+, template concentration.etc. were optimized, and then the specificity and sensitivity of the method were investigated. Results The optimal reaction conditions of RT-LAMP was as follows: 1.6 μL FIP and 1.6 μL FIP (inner primer), 0.5 μL F3 and B3 (outer primer), 2.5 μL2.5 mmol/L dNTP, 2 μL 4 mmol/L MgSO4, 3 μL Buffer, 1.6 μL 10×Bst DNA polymerase, 3 μL DNA templates, 0.5 μL 100×SYBR, adding sterilized double-distilled water to make up 25 μL LAMP reaction system mixtures, and the reaction was under 63 ℃ for 60 min. The results verified that only Cronobacter sakazakii displayed the typical fluorescence curve, and the sensitivity of the real-time fluorescence LAMP for the detection of Cronobacter sakazakii was 8×10-2 CFU/mL. Conclusion The method has the advantages of less time-consuming, strong specificity and high sensitivity, which can provide references for the fast detection of Cronobacter sakazakii in infant formula.