Real-time PCR in analyzing DNA extraction from Cryptosporidium oocysts.

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.