生鲜牛乳总微生物基因组DNA的提取

目的 建立高效快速提取生鲜牛乳中总微生物基因组DNA的方法。方法 以生鲜牛乳为原料, 采用SDS-蛋白酶K法裂解细胞, 酚氯仿有机抽提去除蛋白和醋酸钾溶液沉淀蛋白, 制备样品中总微生物基因组DNA。结果 以NET(Tris?HCl, EDTA, NaCl)作为裂解缓冲液, 蛋白酶K消化得到的基因组DNA纯度和产量较高, 耗时较短; 缓冲液选择NCT(NaCl, CaCl2, Tris?HCl)时, PCR产物特异性低于前者且产量较低。RNA酶消化对产品纯度影响不大且会降低产量。用醋酸钾(KAc)沉淀去除蛋白, 操作快速简单, 耗时最短, 但DNA产量最低。结论 SDS-蛋白酶K法提取的生鲜乳微生物总DNA可以作为牛乳样品进一步检测的分子基础。

Extraction of total microbial genomic DNA in raw milk

Objective To establish an efficient and rapid method for the genomic DNA extraction of microbe from raw milk. Methods Genomic DNA of microbe was prepared after the cell lysis by SDS-proteinase K method and removing protein by phenol-chloroform and potassium acetate. Results Genomic DNA of microbe, which was obtained by using of proteinase K and NET(Tris?HCl, EDTA, NaCl) as buffer, showed some advantages of high purity and yield and time saving.Comparatively, the specificity and yield of polymerase chain reaction(PCR) products were lower while chosing NCT (NaCl, CaCl2, Tris?HCl) as buffer. RNAse hardly affected the purity of DNA, but it lowered the yield of DNA. Protien precipitation with potassium acetate may simplify the operation, but the yield of DNA was the lowest among all the methods. Conclusion Total microbial genomic DNA in raw milk extracted by SDS-proteinase K method can be used as a molecular basis of further molecular biology detection.