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目的建立一种直接用于PCR反应的芽胞杆菌基因组DNA提取的改良方法。方法用十六烷基三甲基溴化铵(Cetyltrimethyl ammonium bromide,CTAB)-溶菌酶-冻融裂解法提取蜡样芽胞杆菌、苏云金芽胞杆菌、纳豆芽胞杆菌基因组DNA,用紫外分光光度计测定提取的基因组DNA在230、260、280 nm波长下的A值,计算DNA浓度,并以提取的基因组DNA为模板进行PCR扩增。结果采用改良CTAB法提取的基因组DNA A260/A280值均在1.8～2.0之间,A260/A230值均大于2.0,DNA浓度均大于110μg/ml;PCR扩增产物均可见1 500 bp的清晰条带,浓度较高,未见其他特异条带。结论 CTAB-溶菌酶-冻融裂解法提取芽胞杆菌基因组DNA简单、高效,并可用于PCR反应,适用于临床分子生物学检验。
Objective To establish an improved method for extracting Bacillus genomic DNA directly for PCR reaction. Methods The genomic DNA of Bacillus cereus, Bacillus thuringiensis and Bacillus natto was extracted with Cetyltrimethyl ammonium bromide (CTAB) - lysozyme - freeze thawing method. The genomic DNA was determined by UV spectrophotometer Extract genomic DNA at 230,260,280 nm wavelength A value, calculate the DNA concentration, and the extracted genomic DNA as a template for PCR amplification. Results The A260 / A280 genomic DNA extracted by modified CTAB method were all in the range of 1.8-2.0. The values of A260 / A230 were all greater than 2.0 and the DNA concentrations were all greater than 110μg / ml. The PCR products showed a clear band of 1 500 bp , Higher concentration, no other specific bands. Conclusion The CTAB-lysozyme-freeze-thaw lysis method is a simple and efficient method for extracting Bacillus genomic DNA and can be used in PCR reaction for clinical molecular biology test.