Establishment of real time fluorescence single primer isothermal amplification for Listeria monocytogenes
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摘要: 以单核细胞增生李斯特氏菌（Listeria monocytogenes,L.monocytogenes）hly A基因为靶序列,设计5’端为RNA序列,3’端为DNA序列的嵌合引物和链终止Blocker序列,经过优化,建立实时荧光单引物等温扩增（Real-time fluorescence SPIA）方法,进行特异性、检出限实验,并对不同DNA提取方法进行比较。结果显示,确定荧光染料SPIA法的最佳反应温度为58℃,反应30 min,引物特异性良好,只有4株不同来源的L.monocytogenes DNA产生典型的S型荧光扩增曲线。对L.monocytogenes纯培养DNA的检出限为3.6×101fg/μL,相应菌液浓度为1.2×101CFU/m L;Realtime fluorescence SPIA检测试剂盒法、水煮法、溶菌酶-蛋白酶K法、饱和酚提取法四种方法提取DNA,均产生典型的扩增曲线,Ct值没有明显差异。对人工添加猪肉火腿样品中的L.monocytogenes,采用水煮法提取DNA的检出限为1.1×102CFU/g。结果表明,所建立的L.monocytogenes的Real-time fluorescence SPIA新型等温扩增方法,特异性强、灵敏度高、不受DNA纯度的影响、快速、简便。
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关键词:
- 单引物等温扩增 /
- 荧光 /
- 单核细胞增生李斯特氏菌 /
- hly A基因
Abstract: In order to establish and apply a real-time fluorescence single primer isothermal amplification ( real-time fluorescence SPIA) for Listeria monocytogenes ( L. monocytogenes) , the RNA/DNA primers and Blockers were designed, which based on hly A gene of L.monocytogenes.The real-time fluorescence SPIA was established to detect the L.monocytogenes after the optimization of the reaction system and reaction conditions, which specificity and detection limit were tested. To detect and compare the DNA of different extraction methods.The results showed that the optimum reaction temperature of fluorescence dye SPIA method was 58 ℃ and the specificity of the primers was good in 30 min, only 4 different sources of L.monocytogenes DNA produced a typical S type fluorescence amplification curve. The detection limit of real-time fluorescent SPIA for L.monocytogenes DNA was 3.6 × 101fg/u L and the concentration of corresponding bacteria was 1.2 × 101CFU/m L in pure culture.The DNA was extracted by four methods, which were kit method, boiling method, lysozyme protease K method, and saturated phenol method were detected by the real-time fluorescence SPIA. There was no significant difference of the four amplification curves Ct value.The detection limit of real-time fluorescent SPIA for L.monocytogenes DNA was 1.1 × 102CFU/g for DNA from pork ham samples was extracted by boiling method.The results demonstrate that the new real-time fluorescence SPIA method for Listeria monocytogenes, which has high specificity and high sensitivity, is not affected by the purity of DNA. -
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