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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%TritonX-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%TritonX-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。
Objective To compare the effects of different methods on the purification of Cryptosporidium oocyst DNA. Methods The isolated and purified Cryptosporidium oocysts were respectively added into the lysis buffer of Promega Corporation (Promega) and Shanghai Jierui Genomic DNA Purification Kit, 2% Triton X-100 and 5% isothiocyanate Guanidine, and combined with freeze-thaw method, protease K method and cleaved cleavage of oocysts, using kit method or Chelex-100 method to purify oocyst genomic DNA, real-time PCR determination of Cryptosporidium oocysts wall protein (COWP) Gene copy number, using Promega kit as a reference to compare the purification effect of different purification methods. Results The lysis of Cryptosporidium oocysts by Promega kit was the best. The COWP gene copy number was (6.45 ~ 9.86) × 106 in the purified oocysts. The others were in the order of lysate [(2.38 ~ 3.69) × 106], 5% guanidine isothiocyanate [(1.27 ~ 21.29) × 105] and 2% TritonX-100 [(2.06 ~ 866.70) × 103]. Cryopreservation method + protease K method + sonication cleavage of Cryptosporidium oocyst DNA best, followed by freeze-thaw + sonication and proteinase K method + sonication method, freeze-thaw method + proteinase K method the worst . Conclusion The combination of freeze-thaw method, protease K method and sonication method can maximize the purification efficiency of oocyst DNA. The DNA of Cryptosporidium oocysts can be purified by using the kit of Jierui Genomic DNA and 5% guanidinium isothiocyanate Get the same results as the Promega kit.