石蜡包埋组织中结核杆菌DNA不同提取方法比较

Comparison of different extraction methods of Mycobacterium tuberculosis DNA from paraffin-embedded tissues

目的探讨结核患者石蜡包埋组织中简易有效的结核杆菌DNA提取方法并进行验证。方法 20份4%甲醛固定石蜡包埋的肺或淋巴结结核组织标本,分别以氯化钠盐析法、酚-氯仿抽提法、一步法、试剂盒法提取其DNA,并通过琼脂糖凝胶电泳、聚合酶链反应(PCR)及膜芯片法分析所提取DNA的质量。结果氯化钠盐析法提取的DNA〔(19.338±6.270)μg〕和一步法提取的DNA〔(20.050±5.591)μg〕最多(P<0.001),一步法的A260/A280比值(1.044±0.137)明显低于其他3种方法(P<0.001);电泳结果显示4种提取方法均得到了基因组DNA;PCR与膜芯片验证显示除一步法外其余3种方法提取的DNA中均检测到结核杆菌DNA。结论氯化钠盐析法提取石蜡包埋组织结核杆菌DNA具有高效简便的特点,是较为理想的方法。

Objective To explore a simple and effective method for the extraction of Mycobacterium tuberculosis DNA from paraffin-embedded tissue.Methods The Mycobacterium tuberculosis DNA was extracted from 20 specimens of 4% formalin-fixed,paraffin-embedded lung or lymph node tissues using salting-out procedure,phenol-chloroform method,one-step method,and genomic DNA purification kit.The quality of DNA was analyzed by agarose gel electrophoresis,polymerase chain reaction（PCR）,and gene membrane chip.Results Salting-out procedure and one-step method yielded more DNA.However,the value of A260/A280 of DNA extracted by one-step method was lower than that of the other three methods.Genomic DNA was obtained by all methods through electrophoresis.PCR and gene membrane chip tests showed that the Mycobacterium tuberculosis DNA was detected in the DNA extracted by all methods except one-step method.Conclusion The salting-out procedure for extraction of Mycobacterium tuberculosis DNA from paraffin-embedded tissue is more efficient and simple.