关键词: 分枝杆菌属, 组织提取物, DNA提取方法

Abstract: Yan Zhenzhen, Jiang Haiqin, Cui Pangen, Wang Hongsheng, Sun Jianfang. Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding authors: Wang Hongsheng, Email: whs33@vip.sina.com; Sun Jianfang, Email: sunjf@163.com 【Abstract】 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 × 105 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 Mycobacteria 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 × 102 cells/ml for all the three methods. With simulated specimens, the detection rate was consistently 100% 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 × 102 cells/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.

Key words: Mycobacterium, Tissue extracts, DNA extraction methods