Gel doctm xr imager

Mycoplasma was assessed for via PCR on cell line DNA derived from cells that had been in culture for a minimum of 2 days. DNA was extracted using QuickExtract^TM (QE) DNA Extraction Solution (Lucigen, Middleton, Wisconsin). The primers are listed in Supplementary Table 6. The PCR reaction was performed in the presence of 5 μl of 5× MangoTaq Buffer + 1.5 μl 25 mM MgCl₂ + 0.5 μl 10 mM dNTPs + 2 μl Myco Fwd + Rev Primers + 0.5 μl Cyto Fwd + Rev Primers + 14.25 μl MQ H₂O + 0.25 μl Mango Taq 5 U/μl by the thermocycler and involved 95 °C for 5 min followed by 45 cycles of: 95 °C for 30 s, 53 °C for 30 s and 72 °C for 30 s. This was followed by one cycle of 72 °C for 5 min and 14 °C hold. The samples were run on a 1.5% agarose gel with Midori Green Advance (Nippon Genetics #MG04) and imaged with the Gel Doc^TM XR Imager (BioRad). Cytochrome B was used as the loading control, to confirm the presence of gDNA in each lane with this band identified at 375 bp. The mycoplasma band was at 520 bp, with a 1 kb ladder used as the reference. All samples were assessed in triplicate.

A conventional PCR assay was used as the reference standard for the LAMP assay. Five primer pairs were selected and tested for their abilities to detect the A27L gene and the F3L gene of MPXV pseudovirus. Optimal primers for A27L (A27L-1F3 and A27L-1B3) and F3L (F3L-1F3 and F3L-1B3) and the reaction temperature (60°C) were identified for conventional PCR assay (see Fig. S1 in the supplemental material). The PCR mixture contained the following components: 12.5 μL of PCR mix reagents (Tiangen Biotech Co., Ltd., Beijing, China), 0.3 μL forward primer (10 μM), 0.3 μL reverse primer (10 μM), 9.9 μL sterile water, and 2 μL DNA template (identical to the volume in the LAMP assay). Amplification was performed on a thermal cycler (Veriti 96-well thermal cycler, Applied Biosystems) with the following cycling profile: denaturation at 95°C for 10 min; 40 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 45 s; and final extension at 72°C for 3 min. Amplified PCR products (5-μL volumes) were separated by 1% agarose gel electrophoresis (120 V, 25 min) and stained with GelGreen. Images were taken using a gel imaging system (GelDoc TM XR+ imager, Bio-Rad Laboratories, Co., Ltd.). Samples positive for the MPXV A27L or F3L gene showed a DNA ladder of ~200 bp.

The serum was collected following centrifugation of clotted peripheral blood at 2,000× g and 4°C for 10 min. M protein/paraprotein were assessed by serum protein electrophoresis (SPEP) using the Hydragel Protein (β1‐β2) 30 Kit (Sebia), according to the manufacturer's instructions. The stained SPEP gels were imaged on a Gel Doc^TM XR+Imager (Bio‐Rad), and the intensity of the paraprotein band/M‐spike was quantitated and normalized to the albumin band using Image Lab Software v6.0.1 (Bio‐Rad).