皮肤分枝杆菌DNA提取方法比较分析

Comparison of three methods for the extraction of mycobacterial DNA

目的比较分枝杆菌DNA提取的常用方法。方法分别采用传统的冻融法和两种试剂盒（A、B）对不同浓度结核分枝杆菌、麻风分枝杆菌和耻垢分枝杆菌纯菌悬液及模拟标本进行总DNA提取。通过检测DNA纯度和PCR产物比较3种方法的提取效果，并应用临床皮肤组织标本进一步验证。结果3种方法提取的分枝杆菌DNA均能用于PCR检测。试剂盒A获得的DNA纯度最高，冻融法其次，试剂盒B杂质最多；灵敏性检测显示3种方法可检测出的纯菌悬液最低浓度皆为102菌细胞／ml；对于模拟标本，在10^3菌细胞／ml时检出率为100％，在10^2菌细胞／ml时试剂盒A、B和冻融法检出率分别为60％（12／20）、55％（11／20）、55％（11／20）；临床标本检测结果提示，试剂盒B可用于石蜡标本DNA的提取，对分枝杆菌感染组织提取率同其它两种方法基本一致。结论试剂盒A通过多次过柱洗涤可快速获得分枝杆菌的优质基因组DNA，为实验研究及临床检测的首选；试剂盒B单次试剂处理除适用于石蜡标本提取DNA外，可用于新鲜组织标本的协同检测。

Objective To compare three methods for the extraction of mycobacterial DNA. Methods Two commercial DNA extraction kits and an ordinary freeze-thawing method were used to extract DNA from the pure suspensions of three species of Mycobacteria （M. tuberculosis, M. leprae and M. smegmatis） at different densities （ 1× 10 to 1 × 10^5 cells/ml）, simulated clinical specimens containing different concentrations of mycobacterial cells （1 ×10 to 1 × 104 cells/ml）. The purity and concentration of the extracted DNA were evaluated. Then, PCR was performed to amplify the 16S rRNA region of Mycobacteria. The performance of the three methods was compared by the purity and concentration of extracted DNA as well as the results of PCR. Further more, 76 clinical skin specimens suspected to be infected with MycoIJacteria were used to further validate the performance of these methods. Results All the extracted DNA samples could be detected by PCR. The highest purity of DNA was obtained by the kit A, followed sequentially by the freeze-thawing method and the kit B. When pure suspensions were used, the detection limit was consistently 1 x 102 cells/ml for all the three methods. With simulated specimens, the detection rate was consistently 100~/o for all the three methods at the concentration of 1 ~ 103 cells/ml, 60% （12/20）, 55% （11/20） and 55% （11/20） for the kit A, kit B and freeze-thawing method respectively at the concentration of 1 x 102 eells/ml. The analysis of clinical specimens showed that the kit B could be used to extract DNA from paraffin-embedded specimens, with the detection rate similar to that of kit A and freeze-thawing method. Conclusions The kit A could rapidly yield high-quality genomic DNA of Mycobacteria by repeated cleaning of columns, and may serve as the optimal method for scientific and clinical studies, and the kit B is suitable for extracting mycobacterial DNA from fresh tissue specimens besides paraffin-embedded specimens.