摘要
目的构建熔解曲线分析法同时检测4种临床常见革兰阳性病原菌,为临床感染性疾病的诊疗提供快速、敏感的分子生物学检测信息。方法选取临床最常见的革兰阳性病原菌:金黄色葡萄球菌、表皮葡萄球菌、屎肠球菌、粪肠球菌做为本课题研究的检测菌种。根据检测菌种的不同,分别选取不同菌种的特异性靶序列,设计种特异性引物。对种特异性引物分别用标准菌株、临床分离目标菌株、临床分离非目标菌株、非细菌性病原体以及人源基因组DNA进行常规PCR扩增以及扩增产物测序,验证引物的特异性。对目标菌种引物进行荧光PCR预实验,筛选Tm值差异较大的多对引物,进行熔解曲线分析法实验,并对熔解曲线法的反应条件与反应体系进行优化。对优化后的熔解曲线法进行特异性验证。通过检洲加入人血液中的不同细菌浓度对该方法进行敏感性分析。应用所构建的方法分别检测临床分离目标菌种各20株,分析每种菌种的熔解曲线图,熔点温度(Tm值)及其标准差(SD)的变化,评价所构建方法的稳定性。结果①对所设计的种特异性引物经常规PCR扩增产物电泳分析显示,目标菌株均有目的特异性扩增产物,非目标菌株、非细菌性病原体以及人源基因组DNA未出现特片性电泳条带。特异性扩增产物测序所得序列比对分析显示与目的菌种序列相符。②对金黄色葡萄球菌、表皮葡萄球菌、屎肠球菌、粪肠球菌的4对引物同时进行熔解曲线分析和mecA基因熔解曲线分析的条什优化结果:预变性温度94℃、120s,变性温度94℃、20s:退火温度53℃、12s:延伸温度68℃、13s;扩增循环数为40个循环,熔解曲线分析温度75℃~95℃。最佳的引物浓度为300nmol/L。③所建立的方法经特异性分析显示,上述4种目标菌种均有特异性熔解曲线,Tm值依次为81.02℃;80.32℃;82.37℃;83.10℃,峰高(cm)/峰宽(cm)均大于2.6,以此为典型的熔解曲线判断标准,则非目标菌、非细菌性病原体以及人源基因组DNA分析未见典型的熔解曲线。④方法的敏感性分析显示,上述4种目标菌和mecA基因可检测到的血液细菌浓度依次为:550、520、470、500与203CFU/mL。⑤对上述4菌和MRSA的临床分离菌各20株进行检测,结果显示,其平均Tm值依次为(80.96±0.688)℃,(80.20±0.729)℃,(82.42±0.599)℃,(83.20±0.713)℃和(80.11±0.15)℃。结论本研究所设计的针对金黄色葡萄球菌、表皮葡萄球菌、屎肠球菌、粪肠球菌和HmecA基因检测引物具有较好的菌种特异性,可用于相应病原菌熔解曲线分析法的建屯;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;但葡萄球菌属内和肠球菌属内的Tm值较接近,必要时,可进一步用单对引物检测分析以鉴定到种。
Objective The objective is to develop methods for detecting clinical common Gram-positive pathogens, which is specific, sensitive and fast for the diagnosis of infectious diseases. Methods The most common clinical Gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis were selected as target bacteria. The species-specific primers were designed according to the specific target gene of different species. The specificity of species-specific primers for target bacteria were validated with conventional PCR by detecting standard strains, clinical isolated target strains, clinical isolated non-target strains, non-bacterial pathogens and human genome DNA. The primers with quite different Tm value were screened by the pre-experimental. The methods for detecting target bacteria by melting curve analysis were constructed. The conditions of melting curve analysis were optimized. The specificity of the methods was validated with the strains as in conventional PCR. The sensitivity of the methods was evaluated with different concentrations of target bacteria in human blood. The stability of the methods were evaluated with 20 clinical isolated target strains respectively by the melting curve, melting temperature(Tm value) and their standard deviation(SD). Results (1) Electrophoresis analysis displayed the specific amplified products obtained from target species, but there were no any products from nontarget strains, non-bacterial pathogens and human genome DNA in conventional PCR. The sequences of the specific amplified products obtained from target species were the same of the selected target gene by Blast in GenBank. (2) The optimized conditions of melting curve analysis were as the follow: an initial denaturation at 94℃ for 120s followed by 40 cycles of DNA denaturation at 94℃ for 20s, primer annealing at 53℃ for 12s, and primer extension at 68℃ for 13s. Melting curve analysis was performed from 75℃ to 95℃. The optimum primer concentration was 300nmol/L. (3) The specificity validation of the methods showed that the specific melting curves obtained from four species of target strains. Tm values of them were 81.02℃, 80.32℃, 82.37℃ and 83.10℃ respectively. The ratios of the height of melting peak(cm)/the width of melting peak (cm) were more than 2.6. Using the ratio as the criterion for determining specific melting curves of the target strain, there were no typical melting curve obtained from nontarget strains, non-bacterial pathogens and human genome DNA. (4) The sensitivity evaluation of the methods showed that 550, 520, 470, 500 and 203CFU/mL of bacteria in human blood could be detected respectively for four species of target stains and MRSA. (5) stability evaluation of the methods displayed that the average Tm values of the four species of target stains and MRSA were 80.96±0.688, 80.20±0.729, 82.42±0.599, 83.20±0.713 and 80.11±0.15 respectively from the clinical isolated target strains. Conclusions The species-specific primers designed in this study were highly specific respectively for four species of target strains and mecA gene, and could be used to establish of the methods for detecting the target species of bacterium by melting curve analysis. The established methods 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. However, Tm values of the bacteria within staphylococcus genus and within the enterococcus genus were very closely. It is necessary to further identify the species of them with single species-specific primer.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2011年第6期449-455,459,共8页
Chinese Journal of Antibiotics
基金
2008昆明市科技计划重大项目(项目号:08S100304)
关键词
熔解曲线分析
方法建立
检测
革兰阳性病原菌
Melting curve analysis
Methods developing
Detection
Gram-positive pathogen