Human genomic dna gdna

NoLimits DNA fragments, YOYO-1 Iodide, and SYTO RNASelect Green Fluorescent Cell Stain were purchased from Thermo Fisher Scientific (Waltham, MA, United States). Human genomic DNA (gDNA) was purchased from Promega Corporation (Madison, WI, United States). Endotoxin-free Dulbecco’s phosphate buffered saline (PBS), molecular grade water, and saponin detergent were obtained from EMD Millipore (Burlington, MA, United States). Tris-EDTA, nuclease-free water was obtained from VWR (Radnor, PA, United States). UltraCruz Blocking Agent and antibodies directed against CD63 (clone MX-49.129.5), TSG101 (clone c-2), and glypican-1 (GPC-1; clone 4D1) were obtained from Santa Cruz Biotechnology (Dallas, TX, United States). Directly conjugated anti-programmed death ligand-1 antibody (PD-L1; clone 2746) was obtained from Biotium (Fremont, CA, United States). Fluorophore conjugated, affinity-purified F(ab)₂ goat anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, United States). K₂EDTA plasma used for spiked gDNA and EV experiments was purchased from Innovative Research (Novi, MI, United States). Quantitative multiplex reference standard gDNA (HD701) was purchased from Horizon Discovery (Cambridge, United Kingdom). Cerebrospinal fluid was obtained from Gemini Bio-Products (West Sacramento, CA, United States).

The MG 23S assay (beta version) (SpeeDx Pty Ltd; Australia) contained all the components for the multiplexed amplification and detection of M. genitalium (MgPa gene) and five mutations in the 23S rRNA gene (A2058G, A2058C, A2058T, A2059G and A2059C) and the internal control. The assay was used according to the manufacturer’s instructions in 20 μL final volume in 96 well plates run on the LC480 II (Roche Diagnostics). Reactions were thermocycled at 95°C for 2 min, followed by 10 cycles of 95°C for 5 s, 61°C for 30 s (-0.5°C per cycle), and 40 cycles of 95°C for 5 s, 52°C for 40 s. Ten-fold serial dilutions of 10⁶ to 10² copies of MgPa and 23S rRNA templates in a background of 10⁴ copies human genomic DNA (gDNA) (Promega) and internal control template were analysed. Control reactions, containing 10⁶ copies of wild type (A2058 and A2059) template in a background of 10⁴ copies human genomic DNA or no-DNA, were also performed. Analysis was performed according to the manufacturer’s instructions.