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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 MgSO_4,3μL 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。
Objective To establish a real-time fluorescence loop-mediated isothermal amplification (RT-LAMP) method for the detection of Crosse bacteria. Methods Primers were designed based on the conserved sequence of 16S ~ 23S rRNA of Corynebacterium krusei (Gene AY702093), and the reaction conditions such as dNTPs, Mg 2+ and template concentration were optimized. The specificity and Sensitivity. Results The best reaction system for real-time fluorescence LAMP assay was 1.6μL FIP and 1.6μL BIP, 0.5μL F3 and 0.5μL B3, 2.5μL 2.5mmol / L dNTP, 2μL 4mmol / L MgSO_4, 3μL Buffer, 1.6μL 10 × Bst DNA Polymerase, 3μL DNA template, 0.5μL 100 × fluorescent dye. Make up 25μL system by sterilized double distilled water and react at 63 ℃ for 60min. No specific fluorescence amplification curve was found for other strains except Cankerobacter sakazakii. Real-time fluorescence LAMP detection Cronobacter sensitivity can reach 8 × 10-2 CFU / mL. Conclusion The method for detecting Cronobacter has the advantages of short time-consuming, high specificity and high sensitivity, which can provide a reference for the rapid detection of Cronobacter in infant formula.