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Cat. No.: ERDDK-50 (for 50T)

Cat. No.: ERDDK-200 (for 200T)

Description

E.coli Residual DNA Detection Kit (qPCR), also known as E.coli Genomic DNA Residue Probe qPCR Kit or E.coli Host Cell Genomic DNA Residue Detection Kit, is a kit designed for the rapid, effective, and highly sensitive quantification of residual genomic DNA from E.coli host cells in various biological product samples using probe-based qPCR (Quantitative PCR). This product is contamination-resistant, containing an optimized ratio of high-quality UDG enzyme and dUTP, which effectively eliminates false positives or low CT values caused by product contamination during PCR amplification.

E.coli is commonly used for large-scale expression and production of target proteins or bioactive molecules, including recombinant proteins, vaccines, and novel medical bioproducts such as anticancer drugs. During the production process of these bioproducts, small amounts of E.coli cell DNA fragments may remain. These fragments can be immunogenic, infectious, or tumorigenic, and high residual levels may trigger immune responses, pathogenic infections, and tumor induction, posing safety concerns. In many countries and regions, drug registration and regulatory agencies have strict requirements and limits on host cell DNA residues in biopharmaceutical products. By detecting and controlling host cell DNA residues, the immunogenicity risk and safety of bioproducts can be assessed. Additionally, for high-throughput sequencing of trace samples, it is necessary to ensure that the enzymes expressed by recombinant E.coli are free from E.coli DNA contamination.

This kit can efficiently and sensitively detect residual DNA from E.coli host cells, with a minimum detection limit as low as femtograms (fg). The kit is based on quantitative PCR (qPCR) for the determination of exogenous DNA residues, using probe-based qPCR with a detection limit of 3 fg/μl and a detection range of 3 fg/μl to 300 pg/μl. The average CT value, average lgCT, and concentration calculated from the standard curve for positive control detection are shown in the table below.

The kit provides the necessary premix for qPCR experiments, Probe qPCR Mix (2X, UDG), and a specific primer-probe mixture. The premix contains high-quality, E.coli DNA-free Taq DNA Polymerase, UDG enzyme, PCR Buffer, dNTPs, dUTP, stabilizers, and magnesium ions, all of which are universal components. The high-purity primer-probe mixture is designed to target the conserved region of the E.coli 23S rDNA sequence, and together they enable ultra-sensitive quantitative detection of E.coli DNA. Additionally, the kit includes E.coli DNA Control as a positive control to verify the kit’s functionality and to construct a standard curve.

This product offers both Low ROX and High ROX options, making it widely compatible with fluorescence quantitative PCR instruments that either do not require ROX or need Low ROX or High ROX as a reference dye. ROX is used to correct fluorescence fluctuations unrelated to PCR, thereby minimizing well-to-well variation. Such variations can be caused by factors like pipetting errors and sample evaporation. Different fluorescence quantitative PCR instruments have different requirements for ROX, so please choose the Probe qPCR Mix (2X) with high concentration ROX (High ROX), low concentration ROX (Low ROX), or without ROX according to the instrument used. Typically, the Probe qPCR Mix (2X, High ROX) can also be used with instruments that do not require ROX or need low concentration ROX.

Components

• Probe qPCR Mix (2X, UDG)
• Primer/Probe Mix (10X)
• Ultrapure Water
• Dilution Buffer
• E.coli DNA Control (30 ng/μl)
• Low ROX (50X)
• High ROX (50X)

Features

• This kit has high specificity; genomic DNA from HeLa cells and CHO cells does not interfere with the detection of E.coli residual DNA. The kit exclusively detects E.coli host cell DNA, with no cross-reactivity with DNA from other species.
• This kit has high stability, and different degrees of fragmented DNA have minimal interference with the detection of E.coli residual DNA. By using varying amounts of Micrococcal Nuclease (MNase) to fragment the E.coli DNA Control to different extents, and then performing qPCR detection with this kit, the results show that DNA fragments longer than 200 bp are generally detected without affecting the detection results. However, if the fragmentation is too severe, resulting in a majority of DNA fragments being shorter than 200 bp, the detected residual DNA amount may be underestimated.
• This kit has good repeatability. The CT values for 12 repeated tests of 3 pg/μl and 30 fg/μl E.coli DNA are shown in the table below, with CV values (coefficient of variation) all less than 5%.​

• This kit can be used in conjunction with Residual DNA Sample Preparation Kit with Magnetic Beads for sample extraction. The recovery rate test results using E.coli DNA Control as a simulated sample are shown in the table below.