Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens

Abstract

This study compared six automated nucleic acid extraction systems and one manual kit for their ability to recover nucleic acids from human nasal wash specimens spiked with five respiratory pathogens, representing Gram-positive bacteria (Streptococcus pyogenes), Gram-negative bacteria (Legionella pneumophila), DNA viruses (adenovirus), segmented RNA viruses (human influenza virus A), and non-segmented RNA viruses (respiratory syncytial virus). The robots and kit evaluated represent major commercially available methods that are capable of simultaneous extraction of DNA and RNA from respiratory specimens, and included platforms based on magnetic-bead technology (KingFisher mL, Biorobot EZ1, easyMAG, KingFisher Flex, and MagNA Pure Compact) or glass fiber filter technology (Biorobot MDX and the manual kit Allprep). All methods yielded extracts free of cross-contamination and RT-PCR inhibition. All automated systems recovered L. pneumophila and adenovirus DNA equivalently. However, the MagNA Pure protocol demonstrated more than 4-fold higher DNA recovery from the S. pyogenes than other methods. The KingFisher mL and easyMAG protocols provided 1- to 3-log wider linearity and extracted 3- to 4-fold more RNA from the human influenza virus and respiratory syncytial virus. These findings suggest that systems differed in nucleic acid recovery, reproducibility, and linearity in a pathogen specific manner.

Comparison of small scale methods for the rapid and efficient extraction of mitochondrial DNA from wheat crop suitable for down-stream processes

“…Varma et al (2007) reported that extraction of large amounts of high-value, high molecular weight DNA can be limited by the presence of large amounts of phenolic compounds, DNases, and organelle DNA. Some DNA extraction methods have numerous potential limitiations, including risks of cross-contaminations because of the large number of steps involved, lower molecular mass of extracted DNA because of degradation, and samples that are unsuitable for high-throughput applications because of the longer extraction time required (Tan and Yiap, 2009;Turci et al, 2011;Yang et al, 2011). In this study, 2 modified methods (cited here as Methods I and II) were compared.…”

Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses

“…The efficiency of both RNA extraction and subsequent amplification can contribute to differences with regard to LOD. Use of different specimen types and RNA extraction protocols significantly influences the performance of the diagnostic procedures (35). In addition, molecular diagnostics of RNA viruses depends on reverse transcription (RT)-conventional PCR or RT-qPCR, a two-step process highly influenced by the choice of reagents (36).…”

Isothermal Recombinase Polymerase Amplification Assay Applied to the Detection of Group B Streptococci in Vaginal/Anal Samples

“…A screening test that could detect women carrying GBS during labor could eliminate the need for prenatal screening at 35-37 weeks and reduce the risk of antibiotic prophylaxis for noncolonized women (3,6 ). The screening test must include a sample preparation method that ensures high recovery of nucleic acids and sufficient purity of clinical samples to control inhibitors (7,8 ). Comparative performance studies of automated extraction platforms have demonstrated the direct correlation between the performance of the extraction system and the imprecision of a molecular assay (7)(8)(9).…”