| 张志凯,海荣,张恩民,俞东征.聚合酶链反应方法检测鼠疫耶尔森菌的内部对照研究[J].中华流行病学杂志,2005,26(1):36-38 |
| 聚合酶链反应方法检测鼠疫耶尔森菌的内部对照研究 |
| Study on the internal control on polymerase chain reaction in Yersinia pestis detection |
| 收稿日期:2003-04-28 出版日期:2014-09-15 |
| DOI： |
| 中文关键词: 鼠疫耶尔森菌 聚合酶链反应 内部对照 |
| 英文关键词: Yer sinia p estis Polymerase chain reaction Internal control |
| 基金项目: |
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| 中文摘要: |
| 目的避免聚合酶链反应(PCR)方法检测鼠疫菌发生假阴性。方法通过克隆,将16SrRNA引物的扩增产物与鼠疫菌F1抗原基因克隆子相连,并以其作为内部对照模板进行PCR试验。结果得到了在包含F1抗原基因中连接有16SrRNA扩增产物的质粒,并初步确立了作为内部对照质粒的参照标准浓度。结论在采用PCR方法检测鼠疫菌时,加入适宜浓度的内部对照质粒作为模板与待检样品同时扩增,可避免假阴性的发生。 |
| 英文摘要: |
| Objective ?? For the detection of Yersinia p estis by polymerase chain r eaction( PCR) , internal control( IC) is required in order to prevent false neg ative r esults that might be caused by PCR inhibitors. Methods ?? F1 antigen w as amplified by PCR w ith primer F1 and the PCR product of primer F1 w ere cloned with TOPO TA cloning Kit. The plasmid of positive clone was then digested w ith Hpa ?? . The digested plasmid and the PCR products of 16S rRNA were ligated w ith T4 DNA ligase befo re the lig ated products were transformed. Isolate plasmid DNA on positiv e clone and its concentration w ere measured. Plasmid DNA on different concentration by PCR amplification w ith primer F1 w as analyzed and the standard concentration of IC was determined. Results ?? Constructing an IC by inserting a 16S rRNA amplico n to the or iginal target DNA between the tw o primer F1 sites, the size was longer than the tar get DNA. T he standard concentration of IC was det ermined. Conclusion?? An optimal IC concentration to increase t he reliability of the PCR assays might be used to prevent false negative results and appear ed to be useful for detection of Yer sinia p estis. |
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