Magnetic Bead-based Extraction Method Research Articles

A novel, magnetic bead-based extraction method for the isolation of antimicrobial resistance genes with a case study in river water in Malawi.

The environment is increasingly recognized as an important reservoir of antimicrobial resistance genes (ARGs), which can be identified using molecular platforms. Yet, environmental surveillance remains an underutilised tool as there is no agreement on the best strategy for sample processing. We aim to develop a low-cost extraction method independent to commercial kits or reagents. We present a novel, magnetic bead-based method for the isolation of ARGs from river water named MagnaExtract. We present this with analytic limit of detection as well as a case study in Southern Malawi. Here we compare the DNA yield from MagnaExtract with commercially available QIAGEN kits and the crude boil and spin method, using a high-resolution melt analysis PCR panel designed for the detection of third-generation cephalosporin and carbapenem-resistant genes from 98 water samples. The MagnaExtract method is comparable, and in some instance's superior to commercially available kits for the isolation of ARGs from river water samples. The MagnaExtract approach offers a simple, affordable, high yielding extraction method that could be used for the detection of ARGs from river water samples in surveillance campaigns in East Africa.

Comparison of methods for the isolation of cell-free DNA from cell culture supernatant.

In vitro characterization of cell-free DNA using two-dimensional cell culture models is emerging as an important step toward an improved understanding of the physical and biological characteristics of cell-free DNA in human biology. However, precise measurement of the cell-free DNA in cell culture medium is highly dependent on the efficacy of the method used for DNA purification, and is often a juncture of experimental confusion. Therefore, in this study, we compared six commercially available cell-free DNA isolation kits for the recovery of cell-free DNA from the cell culture supernatant of a human bone cancer cell line (143B), including two magnetic bead-based manual kits, one automated magnetic bead-based extraction method, and three manual spin-column kits. Based on cell-free DNA quantitation and sizing, using the Qubit dsDNA HS assay and Bioanalyzer HS DNA assay, respectively, the different methods showed significant variability concerning recovery, reproducibility, and size discrimination. These findings highlight the importance of selecting a cell-free DNA extraction method that is appropriate for the aims of a study. For example, mutational analysis of cell-free DNA may be enhanced by a method that favors a high yield or is biased toward the isolation of short cell-free DNA fragments. In contrast, quantitative analysis of cell-free DNA in a comparative setting (e.g. measuring the fluctuation of cell-free DNA levels over time) may require the selection of a cell-free DNA isolation method that forgoes a high recovery for high reproducibility and minimal size bias.

Touch DNA sampling with SceneSafe Fast™ minitapes

To achieve optimal results in the forensic analysis of trace DNA, choosing the right collection technique is crucial. Three common approaches are currently well-established for DNA retrieval from items of clothing, notably cutting, swabbing and tape-lifting. The latter two are non-destructive and therefore preferable on items of value. Even though the most recently established technique of DNA retrieval by adhesive tapes is widely used since quite some years now, little information has been published so far on how well it performs compared to other methods. Even more important, when it comes to choosing the right DNA extraction method for forensic lifting-tapes, the available information one can rely on as a forensic geneticist is quite scarce. In our study we compared the two widely used, commercially available and automation suitable magnetic bead-based extraction methods "iPrep Forensic Kit" and "PrepFiler Express BTA™ Kit" to conventional organic solvent extraction. The results demonstrate that DNA extraction from standardized saliva samples applied to SceneSafe Fast™ minitapes is most efficient with phenol-chloroform. We also provide evidence that SceneSafe Fast™ minitapes perform better than wet cotton swabs in the sampling of touch DNA from cotton fabric. Applying the tape only once in every spot on the tissue is thereby sufficient for a considerably better collection performance of the tapes compared to swabbing.

The research and application of pretreatment method for matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of filamentous fungi

Objective: To establish and evaluate the feasibility of a pretreatment method for matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of filamentous fungi developed by the laboratory. Methods: Three hundred and eighty strains of filamentous fungi from January 2014 to December 2016 were recovered and cultured on sabouraud dextrose agar (SDA) plate at 28 ℃ to mature state. Meanwhile, the fungi were cultured in liquid sabouraud medium with a vertical rotation method recommended by Bruker and a horizontal vibration method developed by the laboratory until adequate amount of colonies were observed. For the strains cultured with the three methods, protein was extracted with modified magnetic bead-based extraction method for mass spectrum identification. Results: For 380 fungi strains, it took 3-10 d to culture with SDA culture method, and the ratio of identification of the species and genus was 47% and 81%, respectively; it took 5-7 d to culture with vertical rotation method, and the ratio of identification of the species and genus was 76% and 94%, respectively; it took 1-2 d to culture with horizontal vibration method, and the ratio of identification of the species and genus was 96% and 99%, respectively. For the comparison between horizontal vibration method and SDA culture method comparison, the difference was statistically significant (χ(2)=39.026, P<0.01); for the comparison between horizontal vibration method and vertical rotation method recommended by Bruker, the difference was statistically significant(χ(2)=11.310, P<0.01). Conclusion: The horizontal vibration method and modified magnetic bead-based extraction method developed by the laboratory is superior to the method recommended by Bruker and SDA culture method in terms of the identification capacity for filamentous fungi, which can be applied in clinic.

Comparative evaluation of two automated systems for nucleic acid extraction of BK virus: NucliSens easyMAG versus BioRobot MDx

The objective of this study was to compare the performance of two automated nucleic acid extraction systems. Specifically, the NucliSens easyMAG system (bioMerieux, Marcy l’Etoile, France), which incorporates magnetic bead technology, was compared with the BioRobot MDx system (Qiagen GmbH, Hilden, Germany), which uses a silica membrane-based method of nucleic acid extraction. Nucleic acids from the BK virus (BKV DNA) were extracted from 98 plasma and 57 urine specimens using the Real-Q BKV quantitation kit (Biosewoom, Seoul, Korea). Failed PCR was defined as negative BKV DNA results having more than 36 threshold cycles of the internal control by the manufacturer's instruction. The PCR failure rate of nucleic acids isolated from plasma samples using the MDx system was similar to that of plasma samples processed using the easyMAG system (2.0% and 3.1%, respectively). The PCR failure rate of nucleic acids isolated from urine samples using the MDx system was higher than that of urine samples processed using the easyMAG system (33.3% and 12.5%, respectively). These data suggest that the PCR inhibitors present in urine specimens are removed more efficiently by the easyMAG system. Among amplified specimens, the discordant results obtained from the two systems revealed that the BKV DNA load ranged from 2.3 log 10 copies/mL to 4.6 log 10 copies/mL. Of the 25 urine specimens that yielded BKV DNA by both extraction systems, 15 specimens (60.0%) yielded higher BKV DNA loads by the easyMAG system, indicating that the easyMAG system extracted nucleic acid more efficiently than did the MDx system. In conclusion, the easyMAG method outperformed the MDx method when used to extract BKV DNA from urine samples. Magnetic bead-based extraction methods such as the easyMAG system are therefore preferable for the quantitation of viral DNA in urine.