Analytical Sensitivity of Helicobacter Multiplex

As positive control, plasmid-DNA was extracted from a dam⁺, dcm⁺E. coli strain containing the selected bacterial target sequences (Eurofins, Ebersberg, Germany) as described before [29 (link)]. The copy number/unit mass was calculated by assuming that 1 bp weighs about 660 Da. Concentration of plasmid-DNA was measured with the NanoDrop 1000. Knowledge of the concentration of the purified DNA preparations allowed computing the number of plasmid/µL that was used to determine the analytical sensitivity of Helicobacter multiplex DNA finder. For internal quality control of mouse DNA, polymerase A gene was co-detected in the Helicobacter multiplex DNA finder.
In all multiplex PCR and hybridization runs, reactions without template DNA were used as assay negative controls indicating reagent contamination.
To check the assay’s specificity, whole genomes of three closely related bacteria (H. canis (ATCC 51402), C. lari (DSM 11375-0313-001), C. coli (ATCC 4994)) as well as Helicobacter-negative murine fecal samples were applied to the Helicobacter multiplex DNA finder.