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[Real-time PCR in analyzing DNA extraction from Cryptosporidium oocysts].
Chen, Sheng-Xia; Wu, Liang; Shen, Yu-Juan; Zhang, Qiu-Xia; Li, Ting-Ting; Jiang, Xu-Gan; Xu, Yu-Xin; Cao, Jian-Ping.
Afiliação
  • Chen SX; 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.
Article em Zh | MEDLINE | ID: mdl-19856501
OBJECTIVE: To compare the quality and quantity of DNA extracted from Cryptosporidium oocysts by different methods. METHODS: Cryptosporidium 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) x 10(6)] than that of Generay commercial DNA kit [(2.38-3.69) x 10(6)], 5% guanidine thiocyanate [(1.27-21.29) x 10(5)] or 2% Triton X-100 [(2.06-866.70) x 10(3)] , 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.
Assuntos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Reação em Cadeia da Polimerase / DNA de Protozoário / Cryptosporidium / Oocistos Limite: Animals Idioma: Zh Revista: Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi Ano de publicação: 2009 Tipo de documento: Article
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Reação em Cadeia da Polimerase / DNA de Protozoário / Cryptosporidium / Oocistos Limite: Animals Idioma: Zh Revista: Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi Ano de publicação: 2009 Tipo de documento: Article