Qiaxcel dna screening gel cartridge

Total RNA isolation and RT‐PCR were performed as described previously.6 Gene expression was analysed relative to the housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh) using the ΔΔCt method. Primers were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, USA) and sequences are available in Table S1. Capillary gel electrophoresis and quantification of PCR products were performed using QIAxcel DNA Screening Gel Cartridge on a QIAxcel Advanced System (Qiagen, Hilden, Germany).

PCR products were sized using QIAxcel DNA Screening gel cartridge (QIAGEN) on the QIAxcel system (QIAGEN, Switzerland), which enabled high-resolution capillary electrophoresis. A 50–800 bp QX DNA Size Marker (Qiagen) was included on QIAxcel runs, and the size of the products was determined using the QIAxcel ScreenGel software (Qiagen). The QIAxcel system produces a digital gel image and an electropherogram for fragment analysis. The expected PCR product sizes of set A were 749bp (HMPV), 534 bp (HADV), 375 bp (HCOV-229E/NL63), 264 bp (HPIV-2), 188 bp HPIV-3), and 139 bp (HPIV-1), while the product sizes of set B were 754 bp (IFV-B), 578 bp (HCOV-OC43/HKU1), 394 bp (HRV-A/B), 273 bp (HRSV-A), 206 bp (IFV-A) and 143 bp (HRSV-B).

Based on this testing, PCRs were performed in a 10 µl reaction mix containing 2.5 µl DNA extract, 0.5 µl BSA (10 mg/ml), 1 µl of primer mix (Table 1), 5 µl KAPA2G Fast Multiplex Mix (Peqlab, Erlangen, Germany) and 1 µl PCR grade water. Cycling conditions on a Mastercycler Nexus (Eppendorf, Germany) were set to 3 min at 95°C, 35 cycles of 15 s at 95°C, 90 s at 62.5°C, 30 s at 72°C and final elongation for 10 min at 72°C. Amplification of all PCR products was verified using the QIAxcel advanced system (version 3.2; Method: AL320; Qiagen) together with the Qiaxcel DNA Screening Gel Cartridge (Qiagen). A successful amplification of each respective target was defined as a DNA fragment of the expected length with a corresponding signal strength of ≥0.07 relative fluorescent units (RFU). Using this diagnostic multiplex PCR assay, each regurgitate sample was subjected to five replicated PCRs.