| TaqMan实时荧光定量PCR检测艰难梭菌 |
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| 引用本文： | 储琼芳,李先平,华玉婷,宋丽琼,肖玉春,黄元铭,朱思逸,任志鸿. TaqMan实时荧光定量PCR检测艰难梭菌[J]. 疾病监测, 2018, 33(5): 417-422. DOI: 10.3784/j.issn.1003-9961.2018.05.015 |
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| 作者姓名： | 储琼芳 李先平 华玉婷 宋丽琼 肖玉春 黄元铭 朱思逸 任志鸿 |
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| 作者单位： | 中国疾病预防控制中心传染病预防控制所, 传染病预防控制国家重点实验室, 北京 102206 |
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| 摘 要： | 目的 建立特异敏感的实时荧光定量PCR方法，用于艰难梭菌的快速检测；评价基于纯培养艰难梭菌和粪便中艰难梭菌的2种标准曲线；并应用该荧光定量PCR法对急性艰难梭菌感染（CDI）的小鼠进行粪便含菌量检测评价。方法 针对艰难梭菌基因组中16S rRNA序列设计特异性引物和探针，建立一套快速检测艰难梭菌含量的实时荧光定量PCR方法，验证方法的特异性、灵敏性；绘制艰难梭菌纯菌浓度梯度稀释标准曲线和粪便中同浓度梯度艰难梭菌的标准曲线，比较两者的差异；用艰难梭菌高毒株NAP1/027感染用抗生素处理的C57BL/6小鼠，建立CDI小鼠模型，同时应用该荧光定量PCR和活菌培养法定量检测小鼠粪便中的艰难梭菌含量变化。结果 建立的TaqMan实时荧光定量PCR具有较高的灵敏性和特异性，生成标准曲线的相关系数为0.999 8，斜率为-3.400 4；用纯培养艰难梭菌和粪便中艰难梭菌分别制备标准曲线，结果表明2种标准曲线定量检测结果差异无统计学意义。建立CDI小鼠模型，应用该荧光PCR能有效、准确的检测出粪便中艰难梭菌的含量，可替代费时费力的活菌培养计数法。结论 用纯培养艰难梭菌来制备标准曲线不影响对含菌粪便标本的准确定量检测，荧光定量PCR能准确快捷地检测CDI小鼠粪便中的艰难梭菌含量，比活菌计数更快速和方便，可用于艰难梭菌感染小鼠模型中小鼠肠道内艰难梭菌定植的定量检测。
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| 关 键 词： | 实时荧光定量PCR 标准曲线 艰难梭菌感染 小鼠模型 |
| 收稿时间： | 2017-10-19 |
Detection of Clostridium difficile with TaqMan-based quantitative RT-PCR |
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| Affiliation: | State Key Laboratory of Communicable Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China |
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| Abstract: | Objective To establish the specific and sensitive real time fluorescence quantitative PCR(RT-PCR)assay for the rapid detection of Clostridium difficile,evaluate two standard curves of quantitative using pure bacterial culture or same concentration of bacteria in feces and quantitatively measure C. difficile counts in feces of mice infected with C. difficile. Methods The specific primers and probe for Taqman based PCR were designed based on 16S rRNA sequence of C. difficile. A TaqMan probe based RT-PCR assay was established,and its specificity,sensitivity and standard curve were assessed. The standard curve based on pure bacterial gradient dilution or the same concentration gradient bacteria in feces were compared to determine the optimal quantitative RT-PCR. Antibiotic-treated C57BL/6 mice were infected with virulent C. difficile strain NAP1/027 to establish C. difficile infected(CDI)mouse model. The amounts of C. difficile in fecal samples of CDI mice were detected by using quantitative RT-PCR and culture method. Results The specificity and sensitivity of this established TaqMan probe based RT-PCR were high. The correlation coefficient and slope value of standard curve were 0.999 8 and-3.400 4 respectively. There was no significant difference in counts of C. difficile in artificial prepared mimic fecal samples using two standard curves of quantitative RT-PCR based on pure bacterial gradient dilution or the same concentration gradient bacteria in feces. Antibiotic cocktail pretreated CDI mouse model was established. The results showed that the quantitative RT-PCR can detect the amount of C. difficile in mice feces with high efficiency and accuracy and can replace the culture method. Conclusion The quantitative RT-PCR is reliable and efficient for the detection of colonization of C. difficile in CDI mouse model by using standard curve based on pure bacteria compared with the culture method. This RT-PCR assay can be used to detect the C. difficile in CDI mouse model. |
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