临床标本细菌基因组DNA提取方法探讨

Improvement of bacterial genomic DNA extraction in clinical specimens

目的优化细菌基因组DNA提取方法,使其适合临床细菌分子生物学检测需要。方法分别采用专用DNA提取液法、热裂解法、溶菌酶法、热裂解法与碱性裂解法组合改良法,对纯培养细菌和临床标本中细菌基因组DNA进行提取。结果专用DNA提取液法、溶菌酶法提取成功率为100%,热裂解法革兰阳性菌提取成功率为0%,革兰阴性菌成功率为100%,碱性裂解液法在NaOH浓度大于4 mmol时提取成功,临床标本在NaOH溶液超过20 mmol/L并含2%SDS时细菌基因组DNA的提取成功率为100%。结论热裂解法与碱性裂解法组合改良法提取细菌基因组DNA方便快速、简单实用,适用临床标本检测。

Objective To optimize bacterial genomic DNA extraction methods of clinical specimens. Methods Different methods of bacterial genomic DNA extraction from pure cultures and clinical specimens were compared. Bacterial DNA Kit, pyrolysis, lysozyme, and the modified method（ pyrolysis alkaline lysis combination method） were used. Results The genomic DNA extraction from pure cultures with Bacterial DNA Kit, lysozyme and combination method were be sufficient for PCR amplification （ 100% ）. However, the success rate of Gram positive bacteria was 0% with the pyrolysis method. Comparing the Bacterial DNA Kit and the pyrolysis alkaline lysis （ NaOH over 20 mmol/L and 2% SDS ） for clinical specimens, we found these two both methods were equal for PCR amplification, and concentrations of NaOH and SDS were key factors. Conclusion Pyrolysis alkaline lysis method was suitable for genomic DNA extraction in clinical specimens.