Bacteria are frequently found as contaminants in cell cultures. Recent studies indicate an overall 4 - 6 % incidence of bacterial contaminations in commonly used cell cultures. Many cell cultures may lack visual signs of bacterial contamination, as assessed by light microscopy or by examination of the culture medium (e.g.tur
bidity, pH indicator). Moreover, it has been demonstrated that standard antibiotics routinely included in cell culture media as a control measure are ineffective against resistant bacterial infections and dramatically alter cell growth, differentiation and metabolism.
Onar® Bacteria is a nucleic acid amplification test based on conventional PCR, for highly sensitive detection of bacterial contamination in various in situ biologicals including cell cultures and virus stocks.
The included Mix contains a primer sets targeting a highly conserved fragment of the 16S rRNA region of bacterial genomes. The expected PCR products have a size of approx. 467 bp (for Micrococcus luteus the band is expected at 447 bp), as visualized on an agarose gel. This allows for detection of a very broad range of bacteria species usually encountered as airborne contaminants in cell cultures, whereas eukaryotic DNA is not amplified. Besides primers and nucleotides, the B
acteria Mix includes a hot-start Taq polymerase and the Internal Control DNA, as an essential tool to monitor the PCR performance. A band at 140 bp on the agarose gel indicates a successful PCR reaction and rules out inhibitory effects of the sample matrix (e.g. culture medium) on the assay itself. The kit contains dUTP instead of dTTP, which can optionally be used in combination with uracil-DNA glycosylase (UNG) (optional: not included in the Onar® Bacteria Kit) to facilitate precursor amplicon degradation and therefore minimize the occurrence of false-positive results.
Applicable in research and industry for direct testing of cell
cultures and biologicals. For QA application, specific validation might
be required. Intended for research use only. Not recommended for
clinical diagnostics or testing of human samples.
Lyophilized Mix: Primers / Nucleotides / Internal Control DNA / Polymerase in aliquots of 25 reactions each.
Lyophilized Positive Control DNA
PCR Grade Water
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Gel electrophoresis at endpoint of PCR
PCR reaction tubes and filter tips
Required Lab Devices
Agarose gel electrophoresis and DNA stain
Shelf Life and Storage
Store the unopened components at 2 °C to 8 °C until the expiry date
indicated on the label. Once rehydrated, the components must be stored
at ≤ -18 °C.
Amplified PCR products are visualized by standard gel
electrophoresis. Amplification of the Internal Control DNA (low
molecular weight band) indicates a successful PCR and the lack of
PCR-inhibiting components in the sample. A band in correspondence of the
target (ca. 460 bp) implies the presence of a bacterial contamination.
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