熔解曲线分析检测临床常见3种革兰阴性菌的方法建立  

Development of the method for detecting three species of clinical common gram-negative pathogens by melting curve analysis

作　　者：陈弟[1] 胡大春[1] 邵剑春[1] 刘德华[1] 周玲[1] 秦海燕[1] 

机构地区：[1]昆明市临床疾病分子生物学重点实验室,昆明市第一人民医院检验科,云南昆明650011

出　　处：《现代预防医学》2012年第23期6259-6261,6263,共4页Modern Preventive Medicine

摘　　要：目的构建熔解曲线分析法同时检测临床常见3种革兰阴性病原菌。方法选取临床常见的铜绿假单胞菌、肺炎克雷伯杆菌、鲍曼不动杆菌作为本课题研究的检测菌种。对目标菌种设计种特异性引物。构建熔解曲线法,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 (1)特异性验证结果显示,目标菌种均有特异性熔解曲线,Tm值依次为88.37℃;86.32℃;80.86℃;(2)敏感性分析结果显示,3种目标菌可检测到的拷贝浓度稀释液依次为:1.34×103拷贝/ml,3.67×103拷贝/ml,3.01×103拷贝/ml;(3)稳定性评价的结果显示,3种目标菌其平均Tm值依次为88.54±0.662;86.38±0.727;80.12±0.607。结论所构建的熔解曲线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。OBJECTIVE To develop method for detecting three species of clinical common Gram-negative pathogens. METHODS The common clinical Gram-negative bacteria including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii were selected as target bacteria. The species-specific primers were designed according to the different species. The method for detecting target bacteria by melting curve analysis was constructed. The specificity of the method was validated with the strains. The solutions with different copies of purified amplification products were analyzed by the method to evaluation its sensitivity. The stability of the method was evaluated with 20 clinical isolated target strains respectively. RESULTS （1） The specificity validation of the method showed that the specific melting curves obtained from three species of target strains. Tm values of them was 88.37℃, 86.32℃, 80.86℃ respectively; （2） The sensitivity evaluation of the method showed that 1.34 × 10^3copies / mL, 3.67×10^3copies / mL, 3.01×10^3copies / mL purified amplification products could be detected respectively for three species of target stains; （3） stability evaluation of the method displayed that the average Tm values of the three species of target stains were 88.54±0.662, 86.38±0.727 and 80.12±0.607 respectively. CONCLUSION The established method in this study has sufficient specificity, sensitivity and stability, and also it is rapid, simple and cheap for detecting the target clinical pathogens and would be useful in clinical microbiology laboratory.

关 键 词：熔解曲线分析 方法建立 检测 革兰阴性病原菌 

分 类 号：R115[医药卫生—公共卫生与预防医学]