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、Mg^2＋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1.6μL FIP和1.6μL BIP,外引物：0.5μL F3和0.5μL B3,2.5μL2.5mmol/L d NTP,2μL 4 mmol/L MgSO4,3μL Buffer,1.6μL 10×Bst DNA聚合酶,3μL DNA模板,0.5μL 100×荧光染料,用灭菌双蒸水补足25μL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 d NTPs, Mg^2＋, 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 d NTP, 2 μL 4 mmol/L MgSO_4, 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.