Residual E Coli DNA Detection qPCR Kit

The Residual E. coli DNA Detection qPCR Kit is designed for the sensitive detection of trace E. coli DNA in a sample using quantitative PCR (polymerase chain reaction). By using a specific primer and probe set that targets the 23S ribosomal RNA gene in E. coli, this kit can achieve a detection limit as low as 5 fg/µl while minimizing background noise. This kit provides enough 2x qPCR Master Mix (no dye) for reactions and includes a positive control, dilution buffer, and ROX reference dye for generating reliable standard curves. Principle of Probe-Based qPCR. Detection utilizes a 6-FAM-labeled probe paired with Black Hole Quencher-1 (BHQ-1). (A) In the intact probe, fluorescence is quenched via Förster resonance energy transfer (FRET). (B) During the extension phase, the 5'-3' nuclease activity of Taq polymerase cleaves the probe, separating the 6-FAM fluorophore from the quencher to generate a fluorescent signal. The absorbance and emission maxima of 6-FAM are 495 nm and 520 nm, respectively. The manufacture of biological drugs, including vaccines, antibodies, and recombinant proteins, is regulated by agencies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) to ensure that clinical drugs do not contain harmful impurities. Most biologicals are prepared using host cell systems such as human cells, insect cells, or bacterial cells, and carry-over of host cell DNA must be kept to a minimum as it could compromise patient safety. Therefore, manufacturers must ensure that the amount of residual host cell DNA is kept within safety levels defined by regulatory agencies. While specific requirements may vary depending on the host cell type and route of administration, these agencies generally recommend a maximum limit of 10 ng of host cell DNA per drug dose. To facilitate quality control, the Residual E. coli DNA Detection qPCR Kit enables the detection of E. coli DNA at concentrations as low as 5 fg/µl, with no cross-reactivity to mammalian cell DNA. The assay targets the bacterial gene encoding the 23S ribosomal RNA, which is present in seven copies per E. coli genome, allowing for highly sensitive detection. Although this gene is conserved among gram-negative bacteria, primer specificity has been validated by BLAST analysis to be exclusive to E. coli.