Comparison of Eight Methods for the Extraction of Bacillus atrophaeus Spore DNA from Eleven Common Interferents and a Common Swab

Abstract

Eight DNA extraction products or methods (Applied Biosystems PrepFiler Forensic DNA Extraction Kit; Bio-Rad Instagene Only, Bio-Rad Instagene & Spin Column Purification; EpiCentre MasterPure DNA & RNA Kit; FujiFilm QuickGene Mini80; Idaho Technologies 1-2-3 Q-Flow Kit; MoBio UltraClean Microbial DNA Isolation Kit; Sigma Extract-N-Amp Plant and Seed Kit) were adapted to facilitate extraction of DNA under BSL3 containment conditions. DNA was extracted from 12 common interferents or sample types, spiked with spores of Bacillus atropheaus. Resulting extracts were tested by real-time PCR. No one method was the best, in terms of DNA extraction, across all sample types. Statistical analysis indicated that the PrepFiler method was the best method from six dry powders (baking, biological washing, milk, plain flour, filler and talcum) and one solid (Underarm deodorant), the UltraClean method was the best from four liquids (aftershave, cola, nutrient broth, vinegar), and the MasterPure method was the best from the swab sample type. The best overall method, in terms of DNA extraction, across all sample types evaluated was the UltraClean method.

A Sample Extraction Method for Faster, More Sensitive PCR-Based Detection of Pathogens in Blood Culture

“…41 Of the three technologies evaluated here, the HMW polysaccharide-based magnetic DNA purification chemistry performed by the Automate Express instrument was the only chemistry capable of generating extracts from culture medium that could be directly used in PCR without any observed PCR inhibition, and the extracts have exceptional 260/280 and 260/230 ratios that allow for accurate DNA quantitation. This technology differed from the other two tested in that it did not rely on lysozyme pretreatment.…”

Comparison of nucleic acid extraction platforms for detection of select biothreat agents for use in clinical resource limited settings

“…While QIAamp was not evaluated in this study, the QG-Mini80 performed acceptably (ranked 6/8 of platforms across the sample types) (Rose et al, 2011). The QG-Mini80 also performed acceptably for extracting detectable nucleic acid within clinically relevant ranges in this study.…”

“…Fujifilm's QG-Mini80 platform for DNA and RNA extraction is a potential alternative to QIAamp for nucleic acid extraction. Rose et al recently described the QG-Mini80, along with the Instagene Only (Bio-Rad, Hercules, CA), as the easiest to use among 8 different methods they evaluated for Bacillus atrophaeus spore DNA extraction (Rose et al, 2011). This technology utilizes similar chaotropic lysis and a thin, silica-based membrane for nucleic acid extraction but, instead of centrifugation, uses positive pressure for sample application, membrane washing, and elution.…”

Rapid Filtration Separation-Based Sample Preparation Method for Bacillus Spores in Powdery and Environmental Matrices

“…While no method proved to be the best for all types of samples, many allowed the detection of B. atrophaeus in the presence of powders. However, all these techniques required incubation times of 10 to 20 min or multiple centrifugation steps (12). The procedures for B. anthracis DNA extraction discussed above are timeconsuming and labor-intensive and require sophisticated laboratory equipment.…”