Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR
Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR
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DOI：
10.1128/jcm.43.5.2471-2473.2005
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发表时间：
2005-05-01
影响因子：
9.4
通讯作者：
Woods, GL
中科院分区：
文献类型：
--
作者：
Aldous, WK;Pounder, JI;Woods, GL
比较从痰液中提取结核分枝杆菌DNA进行定量PCR检测的6种方法：Tris-EDTA （TE）缓冲液、PrepMan Ultra、2%十二烷基硫酸钠(SDS)-10% Triton X有超声和无超声、感染诊断公司（IDI）裂解管和QIAGEN QIAamp DNA迷你试剂盒；都包括15分钟的煮沸步骤。经消化和净化后的痰液中加入结核分枝杆菌ATCC 27294。每种提取方法重复8次。对Smart Cycler和Rotor-Gene 3000进行定量PCR，引物靶向IS6110的83bp片段。使用带有荧光标记的小型grove结合Eclipse探针进行检测。采用内部对照法检测扩增抑制。两种仪器对结核分枝杆菌DNA的检出限为0.5 fg。使用IDI、PrepMan、QIAGEN和TE提取的DNA计算浓度（picogram）在Smart Cycler上分别为42.8、30.4、28.2和7.4，在Rotor-Gene上分别为51.7、20.1、14.9和8.6。用SDS/Triton X进行超声或不超声的提取均被抑制。在所评估的提取方法中，IDI裂解管提供分枝杆菌DNA的最高产量，该过程可以在不到1小时内完成，而QIAGEN提取则需要2.5-3小时。
Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5-3 h for the QIAGEN extraction.