Example of a good run
What are we looking for in a good run?
- Proper Amplification: Smooth curves (Baseline, Exponential Growth, Plateau)
- Negative Control Pass
- Internal Control Pass
- Positive Control Pass (if applicable)
- Red curves indicate internal control amplification.
- Blue curves indicate SARS-CoV-2 target amplification.
Example of a problematic run
No amplification of samples or controls.
Here’s an example of an experiment in which no amplification occurred:
- Red curves indicate internal control amplification.
- Blue curves indicate SARS-CoV-2 target amplification.
If no amplification occurs, here’s what you should do:
- Verify the RNA extraction procedure and control preparation was followed correctly
- Verify the sample setup is correct in the software(SARS-CoV-2 Wastewater Assay)
- Verify the master mix has been stored correctly
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- Rehydatred vial storage conditions: -20 degrees °C
- PCR strip tube storage conditions: Room temperature
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- Check the expiration date of your master mix
- Rehydrate a new vial of master mix, repeat the experiment run
- This can also occur if extracted RNA sat at room temperature and was not immediately mixed with the master mix. Once mixed with the master mix, the PCR sample should sit at room temperature for longer than 5 -10 minutes.
Internal Controls Failed
Internal Controls are intended to confirm that there are no inhibitors present in the reaction and that the master mix is amplifying properly to validate a negative SARS-CoV-2 result. Failed internal controls may indicate the presence of inhibitors.
Here’s an example of an experiment in which the internal controls failed:
- Red curves indicate internal control amplification.
- Blue curves indicate SARS-CoV-2 target amplification.
If your internal controls fail, here’s what you should do:
- Check the internal control of your Negative Control; did it pass?
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- Was the Negative Control prepared correctly? A mixture of master mix (15 uL) and nuclease-free water (5 μL) in a PCR tube
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- Check the expiration date of your master mix
- Verify the master mix has been stored correctly
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- Rehydatred Vial Storage conditions: -20 °C
- PCR strip tube storage condition: Room temperature
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- Verify the RNA extraction and sample procedure were followed correctly.
- These results could be due to residual ethanol in the sample (see Ethanol Inhibition Section)
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- Re-extract samples, ensure all ethanol is removed, and repeat the experiment run
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- Rehydrate a new vial of master mix, repeat the experiment run
Ethanol Inhibition
Residual ethanol in the sample is a common issue leading to failed internal controls. Ethanol is a strong PCR reaction inhibitor; small amounts can interfere with sample results. Here are some best practices to avoid ethanol inhibition:
- As per the Test Kit Instructions, use a pipette to remove excess ethanol and let beads sit for 2 minutes. If there is still ethanol remaining after the 2 minutes have passed, the user can leave the beads to dry for 10-15 minutes to evaporate any remaining ethanol
- Before adding 100 μL of Elution Buffer NA to the magnetic beads, confirm the beads are dry, and that there is no ethanol left on the beads
If inhibition due to residual ethanol occurs, here’s what you should do:
- Re-extract sample and ensure all ethanol is removed before adding elution buffer
- Repeat experiment run
Inhibitors from Wastewater Sample
Inhibitors from the wastewater sample can be carried throughout the extraction process and affect the qPCR reaction causing internal control failure within your sample. This is more likely to occur if the sample is heavily concentrated. Here is how you can overcome residual inhibitors in your sample:
- Verify the RNA extraction procedure was followed correctly
- Dilute extracted RNA with Nuclease-Free water at a 1:1 or 1:5 ratio prior to adding to your master mix. Rerun the qPCR