Comparison of Six Methods of Extracting Mycobacterium tuberculosis DNA from Processed Sputum for Testing by Quantitative Real-Time PCR

Abstract

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.Tuberculosis is a public health problem worldwide, and for optimal control, early diagnosis is necessary (4, 6, 7). Several researchers have developed real-time PCR assays that provide rapid detection of various target sequences of Mycobacterium tuberculosis complex (MTBC) and drug resistance genes in patient specimens (1,3,5,(16)(17)(18)(19). The ability of these assays to detect MTBC in clinical samples is dependent on both the target sequence selected and the efficiency of the DNA extraction procedure. Several methods of mycobacterial cell wall lysis and DNA extraction have been evaluated, including detergents, proteolytic enzymes, mechanical disruption, and temperature changes alone and in various combinations (1, 2, 5, 8-15, 17, 20). The objective of this study was to compare six methods of extracting M. tuberculosis DNA from respiratory specimens: Tris-EDTA (TE) boil extraction (10), PrepMan ultra extraction (Applied Biosystems, Inc., Foster City, CA), Infectio Diagnostics, Inc. (IDI) lysis extraction (Infectio Diagnostics, Inc. Quebec, Canada), QIAGEN QIAmp DNA mini kit (QIAGEN, Inc., Valencia, CA), sodium dodecyl sulfate (SDS)-Triton X extraction (9), and SDS-Triton X plus sonication. The effectiveness of each extraction method was assessed using two quantitative real-time PCR assays.Sample preparation. Digested and decontaminated (Nacetyl-cysteine-2% NaOH) sputum specimens that were culture negative for mycobacteria were pooled for use as the standard respiratory specimen. A suspension of M. tuberculosis ATCC 27294 was prepared in sterile saline and adjusted to the density of a 1.0 McFarland standard. The suspension was diluted 1:10 in saline and used to spike the pooled respiratory sp...

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