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[Real-time PCR in analyzing DNA extraction from Cryptosporidium oocysts]. | LitMetric
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.
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