Objective The objective is to develop method for detecting Escherichia coli, Stenotrophomonas maltophilia. Methods The species-specific primers were designed according to the specific target gene of target stains. The melting curve analysis were constructed. The specificity of the method was validated. The solutions with different copies of purified amplification products were analyzed by the method to evaluation its sensitivity. The stability of the method were evaluated with 20 clinical isolated target strains respectively. Results The specificity validation of the method showed that the specific melting curves obtained from two species of target strains. Tm values of them were 84. 73,82. 84 °C respectively. The sensitivity evaluation of the method showed that 3. 56× 103 ,1. 21 X 101 copies/mL purified amplification products could be detected respectively for two species of target stains. Stability evaluation of the method displayed that their Tm values showed less difference in the same species. Conclusion The established method in this study have sufficient specificity, sensitivity and stability,and also rapidly, simply and cheaply for detecting of the target clinical pathogens and would be useful in clinical microbiology laboratory.%目的 构建熔解曲线分析法检测大肠埃希菌、嗜麦芽单胞菌.方法 针对大肠埃希菌的16S rRNA、嗜麦芽单胞菌的促族酶B亚单位(gyrB)基因设计种特异性引物.构建熔解曲线分析法,并进行特异性验证.检测不同拷贝浓度稀释液进行敏感性分析.检测临床分离目标菌各20株,进行稳定性评价.结果 特异性验证显示,目标检测菌种均有特异性的熔解曲线,融点温度(Tm)值分别为84.73、82.84 ℃.可检测到的拷贝浓度稀释液分别为3.56×103、1.21×104 copy/mL.稳定性评价的结果显示,Tm值(±s)变化范围窄小,差异有统计学意义.结论 所构建方法具有较好的特异性、敏感性和稳定性.并具有快速、简便、成本低廉的特点,可用于临床样本的检测.
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