2/25/2023 0 Comments Texmacs gmp medium![]() Two master mixes were prepared: one to amplify Mycoplasma spp.-specific templates, with the other targeting β-actin. All other reagents used are included with the kit. We were unable to establish a LLOD at or below 10 CFU/mL with the original volumes. This change was necessary to be able to add more input DNA to the reaction, in order to fulfill the 10 CFU/mL sensitivity requirement. PCR was performed using the MycoTOOL Mycoplasma detection amplification kit (Roche) with two modifications: (1) the CHO-specific Gapdh primer set included with the kit was replaced with a human β-actin primer set and (2) the PCR reactions were scaled up from 50 μL to 100 μL total volume. ![]() Pairs of tubes were pooled to generate a total of four vials containing 190 μL of DNA sample in each. 95 μL of dilution reagent was added to each of the eight sample vials and DNA was resuspended by incubating in the thermomixer at 80☌/900 rpm for 15 min, followed by brief vortexing. Supernatants were completely removed by pipetting. ![]() Vials were inverted five times to mix and DNA was pelleted by centrifugation for 3 min at 16,000 × g. 1 mL of washing buffer was used to wash each pellet. Samples were then centrifuged for 3 min at 16,000 × g, and supernatants were removed by pipetting. ![]() 800 μL of precipitation reagent and 2 μL of GlycoBlue coprecipitant (Invitrogen) were added to each vial followed by 20 inversions and vortexing for 5 s. Samples were incubated for 30 min at 56☌/600 rpm in a Thermomixer R with 2.0 mL block (Eppendorf). 50 μL of proteinase K and 700 μL of lysis buffer were added to each vial, followed by vortexing three times for 5-s durations. Each of the 1,900 μL diluted aliquots was split further into four 450 μL aliquots, for a total of eight 450 μL samples. Two 950 μL aliquots of cell sample were diluted with 950 μL of DNA-free water ( Figure S1). High Cell Density DNA Preparation: >5 × 10 6 to 1 × 10 8 Total Cells/mL 190 μL of dilution reagent was added to each of the four sample vials, and DNA was resuspended by incubating in the thermomixer at 80☌/900 rpm for 10 min followed by brief vortexing. 630 μL of precipitation reagent and 2 μL of GlycoBlue coprecipitant (Invitrogen) were added to each vial, followed by 20 inversions and vortexing for 5 s. Samples were incubated for 15 min at 56☌/600 rpm in a Thermomixer R with 2.0 mL block (Eppendorf). 50 μL of proteinase K and 450 μL of lysis buffer were added to each vial followed by vortexing three times for 5-s durations. The successful amplification of Mycoplasma-specific bands in spike-test samples from both sample types established the specificity of the assay by demonstrating that the assay was capable of detecting the target in the presence of multiple matrices.Ĭell samples (≥2 mL) were divided into four aliquots of 450 μL each ( Figure 2). The absence of contamination is confirmed by the lack of positive 400- to 500-bp bands in the Mycoplasma primer PCR negative control reaction (lane 10). These bands represent non-specific amplification products, and they are not the result of contamination of the PCR reactions. Additional bands (>500 bp) are sporadically observed in Mycoplasma amplification reactions ( Figure 3B, M. arginini gel, lane 1). Both species of Mycoplasma were detected in at least two out of three “spike-test” sample aliquots ( Figures 3A and 3B, lanes 3–5), as evidenced by the presence of positive bands in these lanes between 400 and 500 bp. All rounds were successful gels from two representative assays are shown in Figure 3. The specificity of the assay was analyzed in 17 rounds of testing with CAR-T cell in-process and drug product sample types. genomic DNA (gDNA) that are converted to CFU/mL values using empirically derived genome copy to CFU (GC/CFU) ratios. to demonstrate assay sensitivity is avoided through the addition of defined quantities of Mycoplasma spp. nucleic acid via highly sensitive touchdown PCR, and visualization by gel electrophoresis. The protocol involves DNA extraction from samples of cell therapy products, amplification of Mycoplasma spp. Briefly, our Mycoplasma detection assay utilizes the commercially available MycoTOOL PCR Mycoplasma detection kit (Roche) with a modified protocol in order to obtain the required 10 CFU/mL sensitivity level. Herein, we describe a rapid PCR-based assay that we have qualified for use in testing clinical cell therapy products for Mycoplasma spp. is problematic in facilities that generate cell therapy products because it introduces an unnecessary risk of cell product contamination. Gene Editing: Technology & Applications.
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