摘要
目的摸索并明确实时荧光定量PCR技术检测人体肠道粪便中艰难梭状芽孢杆菌的方法。方法用16S rRNA real-time PCR方法检测喘息患儿与正常儿童肠道内梭状芽孢杆菌的数量。结果常规PCR扩增出艰难梭状芽孢杆菌16S rRNA部分基因片段长度为500 bp^750 bp,测序结果正确,符合预期,成功构建了艰难梭状芽孢杆菌实时荧光定量PCR中的标准品,生成的标准曲线r2>0.995;实时荧光定量PCR产物的溶解曲线为单峰,表明实时荧光定量PCR的特异性强。对待测80份样本分批进行荧光定量PCR检测,各组间标准品Ct值的变异系数(CV)为4.96%、3.45%、2.46%、2.43%和2.75%。80份儿童粪便样本中艰难梭状芽孢杆菌含量的对数值为3.70±2.71(copy/g湿便)。结论本研究表明,实时荧光定量PCR方法操作简便、灵敏度高、特异性强、自动化程度高,适用于人体粪便中艰难梭状芽孢杆菌含量的检测。
Objective To explore and identify the method of applying real- time fluorescence quantitative PCR technology for detecting Clostridium difficile in human intestinal tract fecal samples. Methods Evaluate the amount of Clostridium difficile between wheezy children and control group by 16 S rRNA real- time PCR method. Results The length of PCR product fragment which was used to build the standard curve was about 500 bp- 750 bp. The sequencing result was consist with the goals,and the standard sample of Clostridium difficile was successfully established in real- time fluorescence quantitative PCR. The standard curve showed a good linear relationship with r^2 〉0. 995. The primers were specific and the dissolve curve showed single peak. The CV of standards＇ Ct values which calculated from standard were 4. 96%,3. 45%,2. 46%,2. 43% and 2. 75%.The contents of Clostridium difficile in fecal from 80 children was 3. 70 ± 2. 71（ copy / g wet fecal） transformed by logarithmic.Conclusion This study showed that real- time PCR was not only simple,easy operating,but also high specificity,sensitivity and automated. It is suitable for detecting Clostridium difficile in human intestinal tract fecal samples content.
出处
《中国卫生检验杂志》
CAS
2015年第3期305-307,共3页
Chinese Journal of Health Laboratory Technology
基金
国家自然科学基金青年科学基金项目(8130000210-06165)
关键词
实时荧光定量聚合酶链式反应
引物
标准曲线
艰难梭状芽孢杆菌
Real-time fluorescence quantitative polymerase chain reaction
Primer
Standard curve
Clostridium difficile
