Why are other PCR results different from the Co-Dx test?
- Test design: Each test has a unique design which may target different genes or different sections of the organism’s genome. The technology applied in each test may have greater binding afinity, kinetic energy, or characteristics that allow it to detect targets that the other tests can’t detect and vice versa.
- Contamination or inappropriate handling of materials or samples.
- Extraction: Using a different extraction kit between the two tests could lead to unexpected results or quantitative variance, as different sample extraction kits have different levels of performance, sample input volumes, and elution volumes.
- Samples. The sample type or amount used for extraction may contain varying concentrations of nucleic acid and/or specific analyte. For example, due to time of exposure and the natural defense of an organism, not all cells may be infected with a pathogen.
- Elution volume. The elution volume used to resuspend the DNA/RNA at the end of extraction can affect the concentration of nucleic acid. If a greater elution volume is used, then the lower the final concentration of nucleic acid.
- Total reaction volume. Using different total reaction volumes could result in discrepant results. The total reaction volume used for RT-PCR is optimized using specific conditions, machines, and sample types. The total reaction volume established for the test maintains a consistent ratio of sample to reagent.
- Test equivalence. Most commercial tests have been compared to an international standard in order to standardize the results from one to another and they have been reviewed by regulatory bodies. Home brew tests, in many cases, are not compared to international standards and are only subject to the in-house validation methods. Home brew tests also do not require manufacturing under regulated conditions and may vary from lot to lot.
Why are my PCR results negative, when a positive was expected?
- Limit of detection (LoD). The target in the sample could be below the limit of detection, which is the lowest quantity of target organism that can reliably be distinguished from the absence of that substance 95% of the time. When concentrations below the LoD are tested, the results may be less reliable.
- Test design and population. Different tests may target different genes or different sections of the organism’s genome. Some populations may not carry the targetted gene or may have mutations that interfere with the test.
- Sample age. DNA and RNA can degrade over time. Samples that have been stored for long periods may show reduced amplification or the concentration of analyte will be reduced due to degradation.
- Sample integrity– hemolytic sample. When a sample is hemolyzed the DNA can be released from damaged white blood cells causing it to degrade faster or to fragment. Hemolysis of a sample can occur as a result of disease or during sample collection. Samples passed through narrow needles can cause hemolysis.
- Sample integrity- lipemic sample. Lipemic samples contain triglyceride-rich lipoproteins in blood and may result in inability to purify DNA/RNA from proteins, non-homegeniety, or have light scattering effects with increased absorbance in end point reactions. Hemolysis is also enhanced in the presence of lipemia.
- Sample integrity- icteric sample. Icteric samples contain high levels of bilirubin which can interfere with spectral measurements.
- Sample collection types. EDTA collection tubes are the best choice for molecular tests. Heparin should be avoided because it is a PCR inhibitor.
- Extraction. Ensure that the type of extraction matches the analyte being tested. Extraction kits for DNA should not be used when testing for RNA viruses. Conversely, an RNA extraction kit should not be used for DNA targets.
- Pipetting and mixing. We recommend vortexing each kit tube for 3-5 seconds and briefly spinning down to remove residue from lid. This prevents contamination from handling or splashing.
Why are the Ct values for some samples the same, when they have different concentrations of analyte?
This is expected based on the limit of quantification or limit of detection of the assay. LOD is defined as the lowest limit in which the analyte can be reliably detected. The reported LOD for the assay is printed in the instructions for use or product manual and represents the point at which the analyte can be detected 95% of the time. LOQ is the limit of quantification. This is the lowest concentration of analyte that can be quantitated by the assay 95% of the time. The LOD and LOQ can be equal but the LOQ cannot be less than the LOD. The samples between the LOD and LOQ can give the same Ct value, even with different concentrations.
What is the Internal Positive Control (IPC), and why isn’t it amplifying?
- The Internal Positive Control (IPC) is a control that is contained within the test itself. It can be either an endogenous control, which is present in human samples, or an exogenous control, which is spiked into the sample before extraction. It serves two purposes; 1) it verifies that the master mix is working, and 2) it verifies that the extraction worked as intended.
- Failure to amplify could be caused by a failure during the extraction process or the master mix not working as intended, which can be inferred based upon the results of the Positive Control (PC) and the results of other samples.
- Use of sterile samples (culture, CSF, pleural fluids, etc.) may not contain the analyte that the IPC is targeting
- Nucleic acid saturation during the extraction might have occurred due to large volumes of sample matrix or high concentrations of the analyte.
- Inhibitors present in the sample (including ethanol or isopropanol, which are often used in the extraction).
Why is the IPC amplifying in the no template control (NTC)?
- Ct values below 35 could be the result of contamination in either the master mix or nuclease free water. Contamination is easy for the human RNaseP gene, which is present in all humans (including the operator of the test). Special care should be taken to avoid contamination.
- Ct values above 35 could be the result of ubiquitous binding/non-specific amplification or degradation of the master mix components. The degradation could be caused by improper storage, improper handling, or using materials after the expiration date.
Why is there so much variation in the Ct value among standards?
- Variability is inherent in diagnostic testing. According to CAP guidelines, the coefficient of relative variability or coefficient of variation should be as follows:
- CV=<10-15% for mid-high calibrators or concentrations
- CV=20-50% for low calibrators or low concentrations
- Variation outside of the guidelines can be due to operator errors, inadequate mixing, or from the degradation of test components.
Will my sample evaporate without a heated lid?
- It is possible, depending on the type of instrument being used. The CoDx Box uses qPCR tubes that are pre-filled with oil which along with centrifugal rotation of rotor upon start up reduce the likelihood of evaporation per run.
- For balance, we do recommend replacing the balance tubes on a set maintenance schedule due to evaporation from continuous temperature changes per run. See your instrument manual for more details.
How do reagent volumes affect the performance?
- The volumes and LOD or sensitivity are proportionally related. CoDx has optimized the extraction volume sample input, extraction elution volume, and PCR reaction volumes to reduce customer cost, reduce waste or materials, reduce turn- around times, and optimize performance for each assay. Any changes from the instructions or product manual cannot be supported by CoDx and must be validated by the end-user.
What are the volumes required for purchase?
- We work hard to ensure customer satisfaction. Please contact a company representative to discuss the size of order that meets your specific needs.
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