石蜡包埋脱钙骨组织中结核杆菌DNA不同提取方法比较

目的 探讨石蜡包埋的10%硝酸脱钙骨组织中简易有效的结核杆菌DNA提取方法并进行验证.方法 以20例石蜡包埋、10%硝酸脱钙的坏死性肉芽肿炎疑为骨结核的组织标本为研究对象,常规法直接采用德国QIAGEN公司的QIAamp DNA FFPE Tissue Kit方法提取DNA,而改良法中加入QIAamp DNA Micro Kit试剂盒中的carrier RNA,以两种方法分别提取20例标本中结核杆菌DNA,通过荧光定量PCR,比较常规试剂盒提取法和改良提取法对结核杆菌检测结果的影响.结果 改良法提取的DNA[（35.32±3.48）ng/μl]比常规法[（6.68±3.72）ng/μl]提取的DNA显著增多（P＜0.05）,改良法提取的DNA经荧光定量PCR检测结核杆菌的阳性率（55%）明显高于常规法（15%,P＜0.01）.结论 改良法提取石蜡包埋脱钙骨组织中结核杆菌DNA具有高效简便的特点,是较为理想的方法,值得在puncture和极微小石蜡样本中推广.

Comparison of different extraction methods of Mycobacterium tuberculosis DNA in paraffin-embedded decalcified bone tissues

Objective To explore and verify the extraction method of mycobacterium tuberculosis DNA in paraffin-embedded and 10% nitric acid decalcified bone tissues. Methods Twenty paraffin-embedded and 10% nitric acid decalcified specimens of necrotic granuloma suspected as bone tuberculosis were chosen as subjects. QIAamp＇s DNA FFPE Tissue kit was used in the conventional method to extract DNA. While in the modified method, carrier RNA was added into the QIAamp~ DNA Micro kit. The mycobacterium tuberculosis DNAs of the 20 specimens were extracted by the two methods, respectively. Fluorescence quantitative PCR was used to compare the effects of the two methods on the detection resuhs of mycobacterium tuberculosis. Results The amount of DNA extracted by the modified method [ （ 35.32 ± 3.48 ） ng/μl ] was significantly more than that extracted by the conventional method [ （ 6.68 ± 3.72 ） ng/μl, P 〈 0.05 ]. The positive rate of mycobacterium tuberculosis for DNA detected by the modified method （55 % ） was significantly higher than that detected by the conventional method （ 15%, P 〈 0. 05 ）. Conclusion The modified extraction of mycobacterium tuberculosis DNA from paraffin-embedded decalcified bone tissues is more efficient and simple,which is an ideal method and worthwhile to be promoted in the puncture and little paraffin-embedded tissues.