Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections

Abstract

Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals.

Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA

“…Furthermore, addition of a simple RNA purification step, that does not require commercial kits, improved the detection sensitivity of N1-STOP-LAMP to rival RT-qPCR. Our follow-up of five false-negative results (, ), demonstrated the feasibility of including a simple, scalable and rapid magnetic-bead RNA purification step [26], with which we were able to detect SARS-CoV-2 RNA with N1-STOP-LAMP in 4/5 UTM specimens that had previously tested negative by the assay (Table S3). Other opportunities for improvement include the use of specific fluorescent probes to detect amplification products, thus facilitating multiplex reactions and the inclusion of an internal amplification control within each test [24, 34].…”

“…Solid-phase reversible immobilisation (SPRI) on carboxylated paramagnetic beads (Sera-Mag Magnetic SpeedBeads, from GE Healthcare) were prepared for RNA binding as described (https://openwetware.org/wiki/SPRI_bead_mix). RNA purification was performed in 96-well plates with initial lysis by the addition of 25 µl of 6GTD lysis buffer (7.08 g 10 ml −1 guanidine thiocyanate [6M], made up to 8.2 ml with water, 1 ml Tris HCL [pH 8.0] and 800 µl of 1M dithiothreitol), mixed by pipetting ten times and incubation at room temperature for 1 min [26]. To this, 75 µl of 100% ethyl alcohol and 20 µl of prepared SPRI beads were added, mixed by pipetting ten times and incubated at room temperature for 5 min.…”

Validation of a Lysis Buffer Containing 4 M Guanidinium Thiocyanate (GITC)/ Triton X-100 for Extraction of SARS-CoV-2 RNA for COVID-19 Testing: Comparison of Formulated Lysis Buffers Containing 4 to 6 M GITC, Roche External Lysis Buffer and Qiagen RTL Lysis Buffer

“…The 4 M GITC lysis buffer was developed based on knowledge of the role of GITC in RNA extraction and protection (Chomczynski & Sacchi, 1987;Boom et al, 1990), adapted for magnetic glass bead extraction (Hui He et al, 2017), and validated against recognised standards using positive and negative controls. Details of the composition of 4 M GITC lysis buffer and for the preparation of 1 litre are given below.…”

“…The key chemical constituent of the lysis buffer (guanidinium thiocyanate) has become scarce, scientists from the School of Microbiology, University College Cork, combined their long-held knowledge of lysis buffers for RNA extraction, with insights from published work relating to extraction of RNA using magnetic glass beads (Chomczynski & Sacchi, 1987;Hui He et al, 2017). Ultimately, they formulated a lysis buffer, which uses much less guanidinium thiocyanate than standard formulations appear to use (4 M compared to 6 M).…”

Evaluation of Viral RNA Recovery Methods in Vectors by Metagenomic Sequencing

“…The amount of silica magnetic beads used in this study was 25 µL. He and colleagues demonstrated that 20 µL of silica magnetic beads are sufficient to guarantee a high recovery of NAs and also noted that the extraction efficiency reduced when a large amount of silica magnetic beads were added [37].…”