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目的建立实时荧光单引物等温扩增(SPIA)检测阪崎克罗诺杆菌的方法。方法本文以阪崎克罗诺杆菌Omp A基因特异序列为靶序列,设计RNA-DNA组合引物和链终止序列,优化反应体系,对4株不同来源阪崎克罗诺杆菌和21株其他食源性致病菌进行实时荧光SPIA特异性分析,通过不同浓度梯度阪崎克罗诺杆菌菌悬液灵敏度的检测和添加有阪崎克罗诺杆菌的婴儿配方奶粉样品检出限的确定,评价方法的准确度。结果除4株阪崎克罗诺杆菌外,其他细菌均未出现特异性荧光扩增曲线,具有良好的特异性。在40 min内,实时荧光SPIA检测阪崎克罗诺杆菌纯培养基因拷贝灵敏度为每反应1 copies,相应活菌数为1.6×10-1cfu/ml;对婴儿配方奶粉模拟样品中阪崎克罗诺杆菌的检出限是1.5×100cfu/100 g。结论本研究建立的方法具有灵敏度高、特异性强、耗时短、操作简单等优点,适用于实验室快速检测阪崎克罗诺杆菌Omp A基因。
Objective To establish a real-time fluorescence single-primer isothermal amplification (SPIA) method for the detection of Crosse bacteria. Methods Based on the Omp A gene-specific sequence of Corynebacterium sassanii as the target sequence, RNA-DNA primers and chain-termination sequences were designed and the reaction system was optimized. Four different sources of Krasnaka kazakii and 21 other food sources Sex-specific bacteria by real-time fluorescence SPIA specific analysis by different concentrations of Gram-negative bacteria Susceptibility detection of Sakamoto Kasetsu Kasetsu Kaganaka bacteria added to the infant formula milk powder detection limits to determine the method of evaluation Accuracy Results There was no specific fluorescence amplification curve of all the other bacteria except Corynebacterium sinica Kawasaki, which showed good specificity. Real-time fluorescence SPIA detection of the pure culture of Sakanagasaki Kawasakigensi copy sensitivity of 1 copies per reaction, the corresponding viable count of 1.6 × 10-1cfu / ml within 40 min; infant formula milk samples Sakazaki Kro The detection limit of Bacillus is 1.5 × 100cfu / 100g. Conclusion The method established in this study has the advantages of high sensitivity, strong specificity, short time-consuming and easy operation. It is suitable for the rapid detection of Omp A gene in Corynebacterium sassanii in laboratory.