摘要
目的建立荧光标记扩增片段长度多态性(FAFLP)技术平台,探讨鼠疫菌基因分型。方法用5种核酸限制性内切酶消化鼠疫菌基因组DNA,选择最佳内切酶组合,酶切片段经接头连接后,用荧光素标记的5条EcoRⅠ引物和9条MseⅠ引物组成的引物配对扩增,选择最佳引物组合,进行系列PCR条件的优化。扩增产物经ABI PRISM 3100 Genetic Analyzer(遗传分析仪)检测,GeneScan等软件进行分析。结果成功建立FAFLP技术平台。结论FAFLP具有快速、简便、廉价、分辨率高、重复性好和污染少的优点,可用于鼠疫菌的基因分型分析。
Objective To develop an effective method for genotyping Yersinia pestis by fluorescent amplified fragment length polymorphism(FAFLP). Methods Yersinia pestis genomic DNAs were digested with different endonucleases, including ApaⅠ, TaqⅠ, EcoRⅠ, MseⅠ, and HindⅢ, to select optimal endonuclease combination, and then site-specific adapters were ligated, and PCR amplification was carried out with different combinations of five EcoRⅠ adapter-specific primers labelled with fluorescent dye and nine MseⅠ primers. The amplicons were analyzed by using ABI PRISM3100 Genetic Analyzer and then the results were treated with different softwares, such as GeneScan etc. Results FAFLP method of fast, easy and cheap was established. Conclusions Compared with other biomarker analysis methods, FAFLP possesses characteristics with high discriminatory power, reliable reproducibility, and low contamination, which can be used in the study of Yersinia pestis genotyping.
出处
《中国地方病学杂志》
CAS
CSCD
北大核心
2004年第3期258-261,共4页
Chinese Jouranl of Endemiology
