title: 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.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, Utah 84108, USA.
authors.forename: Wade K
authors.initials: WK
authors.lastname: Aldous
authors.forename: June I
authors.initials: JI
authors.lastname: Pounder
authors.forename: Joann L
authors.initials: JL
authors.lastname: Cloud
authors.forename: Gail L
authors.initials: GL
authors.lastname: Woods
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005798:Genes, Bacterial; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D013183:Sputum / Q000737:chemistry / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial