Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 109 After PCR amplification, samples in either strip tubes or 96-well plates can be analyzed directly using the QIAxcel Advanced instrument without further manipulation. Multiple steps of traditional agarose gel separation, including gel casting, PCR product loading, gel electrophoresis, ethidium bromide staining and gel imaging are not necessary when QIAxcel is used. The QIAxcel Advanced system can also perform automated data interpretation by recognizing predefined electrophoretic patterns. After a run, the positive or negative value for each of the eleven possible amplicons in every sample is presented in a report. Materials and methods Primer design The wzx gene, which encodes a flippase required for O-polysaccharide export, was used to design specific primers for serogroups O26, O45, O103, O111 and O145. The wbqE gene, which encodes a putative glycosyl transferase, and wbqF, which encodes a putative acetyl transferase (6), were used to design primers for the detection of serogroup O121. The primers for O157 were designed from the rfbE gene, and primers used for virulence genes were designed and validated in our previous study (4). The 11 pairs of primers were specifically designed to match the majority of available target sequences and to amplify different amplicon sizes that could be easily separated on a gel or on the QIAxcel Advanced system (Table 1). DNA templates Field isolates obtained from cattle fecal samples were stored in CryoCare beads (Key Scientific Products, Stamford, TX) at –80°C. Single colonies were streaked onto blood agar plates (BAP, Remel, Lenexa, KS) and were incubated overnight at 37°C. One or two colonies of each strain were suspended in 1 ml of distilled water and boiled for 10 min. After a short centrifugation, 1 μl of the supernatant was used as the DNA template (approximate amount ranging from 1–100 ng DNA per 25 μl reaction). For cattle fecal sample preparation, approximately 1 g of feces was added to a tube of 9 mL Escherichia coli broth (Difco, ThermoFisher), and cultured at 40°C for 6 hr. One milliliter of the enrichment was transferred to a 1.5 ml tube and centrifuged. The supernatant was Target gene Amplicon size (bp) Target gene Amplicon size (bp) wzxO45 890 wzxO26 417 wzxO103 740 eae 375 stx1 655 rfbEO157 296 wbqEO121 wbqFO121 587 wzxO111 230 wzxO145 523 ehxA 168 stx2 477 Table 1. The molecular targets and their amplicon sizes used in the 11-gene multiplex PCR. Specificity and amplicon sizes were confirmed by sequencing.

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