Why was my sample inhibited?

The COVID-19 Surveillance Test Kit utilizes an endogenous control target to prevent false negatives due to incomplete RNA extractions or unsuccessful PCR reactions. When this target doesn’t amplify in a saliva sample, the sample will return “Inhibited” rather than “Positive” or “Not Detected.” There are a few reasons for why this may occur:

1. Improper sample collection.

   Food debris or other particulates in saliva can interfere with RNA extraction for SARS-CoV-2 and the endogenous control and inhibit the PCR reaction. If the endogenous control is unable to amplify, the sample will return an “Inhibited” result. To prevent inhibition due to improper sample collection, ensure that the subject does not eat, drink, smoke, chew gum, or brush teeth at least 30 minutes prior to saliva specimen collection.

2. Incomplete RNA extraction or heat inactivation.

   The COVID-19 Surveillance Test Kit workflow involves a room temperature extraction step to release target RNA into solution to make it accessible for PCR. Incomplete RNA extraction would limit the availability of the endogenous control gene target. Once the RNA extraction is complete, the extraction enzymes must be inactivated with heat. If these enzymes are not fully inactivated, they may degrade the Common Mix components when the sample is added to the PCR tube and interfere with PCR amplification. To prevent this, add the correct amount of Enzymatic DNA/RNA Buffer 10X to your sample and mix thoroughly. Let the sample incubate for 15 minutes at room temperature (20 °C - 30 °C), mixing the lysate half way through. Then incubate the sample for 10 minutes at 95 °C to inactivate the enzyme completely. 

3. Debris from saliva in sample lysate.

   After the RNA extraction, the sample lysate should be centrifuged to collect any remaining cellular debris from the saliva into a pellet at the bottom of the 1.5 mL tube. The pellet formed includes components that could interfere with the PCR reaction and lead to an “Inhibited” result. To prevent the cellular debris from interfering with the PCR reaction, centrifuge the saliva for a full 20 seconds at a minimum of 3,000 g (RCF). Additionally, disrupting the pellet after centrifugation or allowing the sample to sit at room temperature for an extended amount of time can cause cellular debris to reform back into solution and inhibit the sample. Ensure that the pipette tip doesn’t touch the pellet when pipetting the required amount of sample lysate into your PCR reaction tube.

4. PCR contamination.

   Contamination of consumables or reagents can interfere with the PCR reaction and lead to sample inhibition. Make sure all pipette tips are clean and do not come into contact with any unnecessary surfaces. Change pipette tips between each solution or when pipetting into a new tube. Frequently, wipe down the working area with aqueous detergent such as 5% bleach, including the tabletop, pipettes, and tube racks.

5. Incorrect PCR reaction tube setup.

   Inhibition may occur if the Common Mix is prepared or allocated incorrectly. Ensure that the correct amount of volume for each Common Mix component is added to a new 1.5 mL tube and homogenize before pipetting the correct amount into your PCR reaction tubes.