| 采用实时PCR技术比较不同方法提纯 |
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| 引用本文： | 陈盛霞,吴亮,沈玉娟,章秋霞,李婷婷,姜旭淦,徐馀信,曹建平.采用实时PCR技术比较不同方法提纯[J].中国寄生虫学与寄生虫病杂志,2009,27(2):130-134. |
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| 作者姓名： | 陈盛霞 吴亮 沈玉娟 章秋霞 李婷婷 姜旭淦 徐馀信 曹建平 |
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| 作者单位： | 1 中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,世界卫生组织疟疾、血吸虫病和丝虫病合作中心,上海 200025; 2 江苏大学基础医学与医学技术学院,镇江 212013; |
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| 摘 要： | 目的 比较不同方法提纯隐孢子虫卵囊DNA的效果。 方法 将分离纯化的隐孢子虫卵囊分别加入美国Promega公司（简称Promega）和上海捷瑞公司（简称捷瑞）基因组DNA纯化试剂盒的裂解液、2% Triton X-100和5% 异硫氰酸胍,同时联合使用冻融法、蛋白酶K法和声裂法裂解卵囊,用试剂盒法或Chelex-100法提纯卵囊基因组DNA,用实时PCR测定隐孢子虫卵囊囊壁蛋白（COWP）基因拷贝数,以Promega试剂盒为参照比较不同纯化方法的提纯效果。 结果 Promega试剂盒的裂解液裂解隐孢子虫卵囊的效果最好,纯化所得卵囊COWP基因拷贝数可达（6.45～9.86）×106;其他依次为捷瑞试剂盒的裂解液（2.38～3.69）×106]、5%异硫氰酸胍（1.27～21.29）×105]和2% Triton X-100（2.06～866.70）×103]。冻融法+蛋白酶K法+声裂法提纯隐孢子虫卵囊DNA效果最佳,其次为冻融法+声裂法和蛋白酶K法+声裂法,冻融法+蛋白酶K法效果最差。 结论 冻融法+蛋白酶K法+声裂法联合使用能使卵囊DNA的提纯效率达到最大,捷瑞基因组DNA纯化试剂盒和5%异硫氰酸胍裂解法提纯隐孢子虫卵囊DNA可获得与Promega试剂盒相近的效果。
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| 关 键 词： | 隐孢子虫 卵囊 实时PCR DNA 提纯 |
Real-time PCR in Analyzing DNA Extraction from Cryptosporidium Oocysts |
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| CHEN Sheng-xia,WU Liang,SHEN Yu-juan,ZHANG Qiu-xia,LI Ting-ting,JIANG Xu-gan,XU Yu-xin,CAO Jian-ping.Real-time PCR in Analyzing DNA Extraction from Cryptosporidium Oocysts[J].Chinese Journal of Parasitology and Parasitic Diseases,2009,27(2):130-134. |
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| Authors: | CHEN Sheng-xia WU Liang SHEN Yu-juan ZHANG Qiu-xia LI Ting-ting JIANG Xu-gan XU Yu-xin CAO Jian-ping |
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| Affiliation: | 1 National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention,Key Laboratory of Parasite and Vector Biology,MOH,WHO Collaborating Centre for Malaria, Schistosomiasis and Filariasis,Shanghai 200025,China;2 School of Medical Science and Laboratory Medicine,Jiangsu University,Zhenjiang 212013,China |
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| Abstract: | Objective To compare the quality and quantity of DNA extracted from Cryptosporidium oocysts by different methods. Methods Cryptospordium oocysts were treated with different kinds of lysis buffers from USA Promega（Promega）and Shanghai Generay（Generay）commercial DNA extraction kits,2% Triton X-100 and 5% guanidine thiocyanate. The oocysts were then broken down by freeze-thawing,proteinase K and sonication. Genomic DNA was purified using the commercial kits or Chelex-100. Real-time PCR technique was used to determine the copies of Cryptosporidium oocyst wall protein（COWP）gene. The Promega commercial DNA extraction kit was used as control. Results The Promega kit resulted in a higher copy number of COWP gene（6.45-9.86）×106]than that of Generay commercial DNA kit （2.38-3.69）×106 ], 5% guanidine thiocyanate（1.27-21.29）×105] or 2% Triton X-100 （2.06-866.70）×103], respectively. The method of freeze-thawing plus proteinase K plus sonication provided the highest copy number of COWP gene. Conclusion The method of freeze-thawing + proteinase K + sonication is most effective. The effect of DNA extraction by Generay kit and 5% guanidine thiocyanate is similar to that of Promega kit. |
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| Keywords: | Cryptosporidium Oocyst Real-time PCR DNA Cryptosporidium" target="_blank">Extraction')">Cryptosporidium Oocyst Real-time PCR DNA Extraction |
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