Evaluation of Automated and Manual Commercial DNA Extraction Methods for Recovery of Brucella DNA from Suspensions and Spiked Swabs

Abstract

This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean microbial DNA isolation kit. These six extraction methods were performed upon three pathogenic Brucella species: B. abortus, B. melitensis, and B. suis. Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent Brucella spp. Real-time PCR analysis using Brucella genus-and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure kit and MagNA Pure Compact performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover Brucella DNA from PBS bacterial suspensions and from swab specimens and, thus, that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for Brucella.

Evaluation of DNA Extraction Methods Suitable for PCR-based Detection and Genotyping ofClostridium botulinum

“…The effectiveness of the molecular methods are strongly influenced by the DNA extractionin fact, it is widely accepted that DNA extraction can influence the sensitivity of PCR-based methods for levels of DNA yield, purity, and integrity. [30][31][32] Lower purity of nucleic acids due to the PCR inhibitors may result in inefficient PCR amplification. PCR inhibitors may denature or precipitate DNA or bind magnesium ions, reducing the enzymatic activity of DNA polymerases.…”

Current Methods of Human and Animal Brucellosis Diagnostics

“…The first crucial step of PCR based methods is DNA isolation from biological samples, since its quality has a significant impact on the sensitivity of the method [13,14]. Initially, PCR based identification has been developed for the determination of bacterial isolates [15], but now these methods are also used for detection of Brucella species in clinical samples of human and animals [13].…”

Swab Protocol for Rapid Laboratory Diagnosis of Cutaneous Anthrax

“…Manual DNA purifications were performed with the QIAamp DNA blood minikit (Qiagen, Inc., Valencia, CA), which utilizes silica spin-filter technology for DNA purification. The procedures were carried out according to the manufacturer's instructions (8). The isolated DNA samples were filtered through 0.1-m centrifugal filter units (Millipore Corporation) as described previously (7) and stored at Ϫ20°C throughout this study.…”

“…Automated DNA purifications were performed using the MagNA Pure Compact and MagNA Pure LC instruments with the MagNA Pure Compact nucleic acid isolation kit I and MagNA Pure LC DNA isolation kit I (Roche), respectively. Both instruments utilize magnetic bead technology for DNA purification, and the procedures for their use have been described previously (8). Manual DNA purifications were performed with the QIAamp DNA blood minikit (Qiagen, Inc., Valencia, CA), which utilizes silica spin-filter technology for DNA purification.…”