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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 and validate the simple and effective DNA extraction method of Mycobacterium tuberculosis in paraffin-embedded tissues of patients with tuberculosis. Methods Twenty specimens of paraffin-embedded lung or lymph node tissue were fixed in 4% formalin. The DNA was extracted by sodium chloride salting-out method, phenol-chloroform extraction method, one-step method and kit method respectively. The DNA was extracted by agarose gel Electrophoresis, polymerase chain reaction (PCR) and membrane chip method to analyze the quality of DNA extracted. Results The DNA extracted by sodium chloride salinity 〔(19.338 ± 6.270) μg〕 and DNA extracted by one step 〔(20.050 ± 5.591) μg〕 had the highest (P <0.001) and A260 / A280 ratio ) Were significantly lower than those of the other three methods (P <0.001). The results of electrophoresis showed that genomic DNA was obtained from all of the four extraction methods. PCR and membrane microarray showed that all of the DNA extracted from the three methods detected M. tuberculosis DNA. Conclusion NaCl salting-out method to extract paraffin-embedded Mycobacterium tuberculosis DNA is characterized by high efficiency and convenience, which is an ideal method.