阪崎克罗诺杆菌TaqMan-MGB探针实时荧光PCR快速检测技术研究
Rapid Detection of Enterobacter Sakazakii with TaqMan-MGB Probe Based on Real-Time PCR Assay
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摘要:
目的 建立阪崎克罗诺杆菌TaqMan-MGB探针实时荧光定量聚合酶链反应(RT-PCR)快速检测方法。 方法 在阪崎克罗诺杆菌保守序列设计一对特异性引物和TaqMan-MGB探针,通过对PCR反应条件和扩增体系的优化,建立阪崎克罗诺杆菌TaqMan-MGB探针实时荧光PCR检测方法;用已知浓度的阪崎克罗诺杆菌验证其敏感性;用阪崎克罗诺杆菌、大肠埃希菌、鸭沙门菌株、柠檬酸杆菌等菌株验证其特异性;用婴幼儿配方粉等食品开展TaqMan-MGB探针实时荧光PCR技术与常规检测方法符合性比较。 结果 本研究建立的阪崎克罗诺杆菌实时荧光PCR检测技术,反应循环(cycle threshold, CT)值与模板浓度的对数值具有良好的对应关系& #374;=-3.06& #215;log(X)+36.63, R2=0.999,最低检测浓度为100 CFU/mL,经36℃增菌8 h最低检测限可达1 CFU/mL,敏感性较普通TaqMan探针高10倍; 11株阪崎克罗诺杆菌CT值均小于30,而大肠埃希菌、鲍曼不动杆菌、柠檬酸杆菌等30株菌株CT值大于30或扩增曲线成一平滑直线;隔天连续5次反应CT值变异系数小于5%; 140件不同批次不同婴幼儿配方粉、192件婴幼儿谷物辅助食品检测结果与常规分离培养比较,差异无统计学意义(χ2=0.624, P>0.05)。 结论 阪崎克罗诺杆菌TaqMan-MGB探针实时荧光PCR快速检测技术具有特异性强、敏感性高、易操作等优点,适用于婴儿配方粉等食品开展阪崎克罗诺杆菌快速检测与监测工作。 Abstract:Objective To establish a method using TaqMan-MGB probe and fluorescence real-time polymerase chain reaction (RT-PCR) for rapid detection of Enterobacter sakazakii. Methods A pair of specific primers and one TaqMan-MGB probe were designed based on the nucleic acid sequence of rpsU gene. After optimization of PCR reaction conditions and amplification system, a TaqMan-MGB probe real-time fluorescence PCR method for detection of Enterobacter sakazakii was developed. The sensibility of this method was evaluated by using solutions containing Enterobacter sakazakii strains with known concentration, and the specificity was evaluated using strains such as Enterobacter sakazakii, Escherichia coli, and Salmonella enterica. Meanwhile, comparison between this protocol and the conventional detection method was performed by using infant formula powder. Results High corresponding relationship (R2=0.999) between the CT values and the logarithm of bacterial concentrations was presented in this proposed detection method. The minimum detectable concentration of Enterobacter sakazakii attained 1000CFU/mL. After enrichment for 8 h at 36℃, the lowest detection limit was 1 CFU/mL. The CT values of all 11 strains of Enterobacter sakazakii were less than 30, whereas the CT values of 30 other bacteria strains including Escherichia coli, Acinetobacter baumannii, and Citrobacter were all higher than 30, and the related amplification curves were even smooth straight lines. The CT values remain stable and the coefficient of variation was lower than 5% after five other-day back-to-back tests. There were no significant differences between the results detected by this proposed assay and by conventional isolation and culture in the test results of 140 different batches of infant formula powder and 192 items of infant cereal food supplement (χ2=0.624, P>0.05). Conclusion The strengths of the TaqMan-MGB probe realtime fluorescence PCR method for detection of Enterobacter sakazakii include high specificity, high sensitivity, easy to operate, and therefore it is suitable for the rapid detection of Enterobacter sakazakii in infant formula milk powder and other food.
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