摘要
目的采用3种方法提取微小隐孢子虫卵囊DNA,并用于PCR检测以进行比较。方法微小隐孢子虫卵囊经多次冻融加热破壁后,采用螯合树脂(Chelex-100)、酚/氯仿和基因组DNA纯化系统试剂盒3种方法提取微小隐孢子虫卵囊DNA,并根据微小隐孢子虫基因序列(L16996)设计一对寡核苷酸引物,分别对3种方法制备模板进行PCR扩增分析。Chelex-100提取的DNA也用于观察PCR检测的敏感性。结果3种方法制备的微小隐孢子虫卵囊模板用于PCR检测均获得1条446 bp条带,Chelex-100提取的DNA用于PCR检测的敏感性至少达0.5个卵囊。结论3种方法提取的微小隐孢子虫卵囊DNA均可用于PCR检测,Chelex-100法是一种高效而快速的微量提取DNA方法,适用于对隐孢子虫DNA的检测。
Objective To establish three methods of DNA extraction from Cryptosporidium parvum oocysts and test by PCR. Methods After three freeze-thaw cycles, three kinds of templates were extracted from the oocysts by Chelex- 100, phenol/chloroform or genomic DNA purification system kit, and used for PCR detection. According to the sequence of a C. parvum gene （L16996）, a pair of primers was designed and synthesized, and used for PCR. The sensitivity of the template by Chelex-100 mcthod was also tested by PCR. Results One 446 bp PCR product was observed by agarose gel electrophoresis for all three kinds of templates. The PCR sensitivity by Chelex-100 extracted DNA reached for detection of a specimen containing only 1/2 oocyst. Conclusion The three kinds of extraction can all be served as templates for PCR detection of C. parvum oocysts, while Chelex- 100 method is simpler, quicker and more reliable for DNA extraction of the parasite.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2005年第4期228-230,共3页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家"十五"科技攻关(No.2003BA712A03-06)~~
