The benefit of the artificial qPCR Extraction Control cells in evaluation of the extraction process is the possibility to validate sample extraction process. Signal derived from the qPCR Extraction Control confirms the success of the extraction step and monitors co-purification of PCR inhibitors that may cause biased or PCR false negative results. Housekeeping genes or spike in controls do not monitor the whole extraction process, especially the lysis step and in the case of housekeeping genes, low extraction efficiency and degraded samples.

When performing DNA extraction, it is often advantageous to have an exogenous source of DNA template that is spiked into the sample prior to lysis. This control DNA is then co-purified with the sample DNA and can be detected as a PCR positive control for the whole extraction process.

Although it is not mandatory yet, regulators across the world are tightening up recommendations for the addition of internal controls, EU: IVDD to IVDR 2017/746 “Control materials with quantitative or qualitative assigned values intended for one specific analyte or multiple analytes shall be classified in the same class as the device.” MDSAP with FDA (“general controls ensuring effectiveness of the device”), TGA (“provisions for the user, at time of use, of device performance”), MHLW and ANVISA

Yes, it is possible that some of the genes will be the similar, the genotype of the E. coli that we use is F- deoR endA1 recA1 relA1 gyrA96 hsdR17(rk-, mk+) supE44 thi-1 phoA Δ(lacZYA-argF)U169 Φ80lacZΔM15 λ pBR322 (ranseqb1 AmpR). You will need to check before using the qPCR Extraction Control with other bacteria.

The primers and probe for the qPCR Extraction Controls are in the Control Mix.

We recommend using the probe-based detection method for quantification of DNA extraction controls, but it would be possible to use other PCR applications for detection of the extraction controls, such as an intercalating dye such as SYBR^® Green (but this would be singleplex), or endpoint PCR.

The two colors are for the fluorescent dyes on the qPCR Extraction Control probe. Control Mix Red (Cy5 – emission wavelength = 670nm) and Control Mix Orange (HEX – emission wavelength = 560nm). This allows you to choose one that will fit with your existing protocol in a multiplex qPCR assay.

No, the qPCR Extraction Control will work with any commercially available DNA extraction method, such as silica-membrane extraction kits, magnetic bead-based extraction, phenol-based extraction and CHELEX matrices and has been tested on a wide range of qPCR platforms.