qPCR Extraction Controls Monitor extraction and co-purificaton of inhibitors with a single control
Molecular IVD companies continue to experience tighter and tighter regulations in regard to incorporating appropriate positive and negative controls into their assays. A common practice for qPCR assays is to spike a known amount of control DNA into the sample after DNA extraction. Adding control DNA after extraction allows for monitoring of inhibition within the assay but has no value as an extraction control. The ideal situation is to have the test sample and internal control undergo exactly the same processing prior to qPCR.
• Extraction control undergoes the same processing as test sample • Can be used with a variety of sample types including inhibitor-rich samples such as blood, urine and sputum • Confirms successes of extraction step and monitors co-purification of PCR inhibitors
• Probe based designed specifically for multiplex qPCR assays • Control DNA is unique with no homology to any organism
Meridian has developed novel extraction controls for qPCR diagnostic assays that are able to more closely mimic the test sample, compared with traditional spike controls. This provides the advantage of monitoring both the success of the extraction step and the presence of any co-purified inhibitors both of which could affect the assay results. The qPCR Extraction Controls use E. coli cells of a known concentration that contain the Internal Control DNA sequence (with no known homology to any organism, so that it does not interfere with the detection of the sample target DNA). Genetic material from the test sample and the DNA extraction control are simultaneously extracted by common extraction methods, with the control being as sensitive to inhibitors and extraction failure as the test sample.
qPCR Extraction Control Red
qPCR Extraction Control Orange
The qPCR Extraction Control is added to the lysis buffer along with the test sample before extraction to monitor poor extraction yield, PCR inhibition, incorrect pipetting or cycling parameters.
Control mix (includes primer and probes for control) is added to the reaction mixture
LYSIS & EXTRACTION
qPCR Extraction Control
Possible Result Target
Internal Control DNA Interpretation
Target(s) and internal control DNA detected.
Target(s) not detected, internal control DNA detected, indicates a sucessful extraction and qPCR reaction. Invalid result: Target(s) and internal control DNA not detected, repeat test.
qPCR extraction control starts monitoring from lysis and extraction
Invalid result: Internal control not detected, repeat test.
qPCR Extraction Control Performs Identical To Spiked Internal Control DNA In Monitoring Inhibition
A) Spiked Internal Control DNA (green channel) and B) qPCR Extraction Control (red channel) were amplified from HEK293 cells. A standard column based genomic DNA extraction kit was used, with the lysis buffer or binding buffer being substituted with PBS to simulate inefficient extraction. Conditions were 10 min at 95 °C 10 s, 60 °C 30 s. Complete lysis step (red) and the pattern on inhibition for no lysis (orange) and no binding buffer (green) are the same across both types of controls.
Suitable For Multiplex Or Singleplex Reactions
A) A fragment of the beta 2 microglobulin (ß2MG) gene and B) A fragment of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified from HEK293 cells (green channel) and C) the internal control sequence were amplified from the qPCR Exctraction Control. Conditions were 10 min at 95 °C followed by 50 cycles 95 °C 10 s, 60 °C 30 s. The results illustrate that there is no difference in Cts between singleplex and multiplex reactions.
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