Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 131 Materials and Methods Samples were collected and total RNA/DNA was extracted using the QIAamp® Viral RNA Mini Kit. A two-tube multiplex reverse-transcription PCR assay (two-tube assay) was used to detect 16 respiratory viruses based on their amplicon size differences. Nucleic acid purification DNA extraction was performed using the Wizard Plus Miniprep® DNA Purification System (Promega) according to the Swiss Food Manual (4). DNA was extracted from 200 mg ground meat from bison, chamois, crocodile, duck, emu, kangaroo, kudu, ostrich, quail, rabbit, red deer, roe deer, springbok, and water buffalo and was eluted in 50 µl of elution buffer according to the manual. PCR-RFLP analysis Primers to conserved regions of the vertebrate mitochondrial cytb gene were used to amplify a 359 bp fragment (1, 3, 5). PCR amplification of 25 µl reactions, as well as restriction analysis, was performed as described in (5). PCR- RFLP products were separated on 3% agarose gels in TAE buffer, and the BenchTop 100 bp DNA Ladder (Promega) was included in the analysis. In addition, analysis was performed on the QIAxcel system using the QIAxcel DNA High Resolution Kit with the OM700 method. The QX  Alignment Marker 15 bp/1 kb was included in the analysis. Results Using a set of endonucleases, all species could be identified with both conventional agarose gel electrophoresis and the QIAxcel capillary electrophoresis system. Using the QIAxcel system, 24 samples were analyzed in approximately 30  minutes. Analysis using agarose gel electrophoresis, which involves more steps for handling and documentation, required at least three times as long. Representative analysis results are shown in Figures 1 and 2. Fragments shorter than 100 bp were not visible on the agarose gel. A 35 bp fragment from samples 11 and 12 (Figure 1) and fragments between 50 bp and 80 bp from samples 11, 12, 14, and 15 (Figure 2) were detected using the QIAxcel system but not with agarose gel electrophoresis. A B Figure 1. PCR-RFLP analysis (RsaI) of meat from several exotic and game species. DNA isolated from crocodile (1), kangaroo (2), ostrich (3), duck (4), red deer (5), roe deer (6), kudu (7), springbok (8), quail (9), rabbit (10), bison (11, 12); water buffalo (13), chamois (14), and emu (15) was amplified by PCR, digested with RsaI and analyzed using A. the QIAxcel system with the QIAxcel DNA High Resolution Kit or B. conventional agarose gel electrophoresis. M: 100 bp DNA Ladder. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 15 M 12345678910111213141512345678910111213141515

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