Why is my Positive Control invalid?

The COVID-19 Surveillance Test Kit utilizes a Positive Control to verify successful PCR reaction setup and target detection. The Positive Control includes an endogenous control target as well as a SARS-CoV-2 gene target. Lack of PCR amplification for the Positive Control may indicate that the PCR reaction was incomplete or inhibited. Here are some potential reasons for an invalid positive control:

  1. Using the incorrect cycling protocol

    Choosing to “Run a Test Kit” in the Open qPCR user interface for the COVID-19 Surveillance Test Kit allows you to bypass the cycling protocol setup and sample/target assignment. If using another compatible qPCR thermocycler, refer to the manual to ensure that you are manually setting up the correct cycling conditions and that the proper data collection step is selected. 
  2. Errors in Common Mix preparation

    The Common Mix is a solution that facilitates the PCR reactions and includes Sahara One-Step RT-qPCR Master Mix with UNG, COVID-19 Surveillance Oligo Mix, and COVID-19 Surveillance Cofactor Buffer. The required volume of each component will vary depending on how many reactions (number of controls plus the number of samples) you are running. Refer to the manual for the amount of each component to add and ensure that the combined solution is thoroughly homogenized and briefly spun down. Ensure that the required volume for each PCR reaction is pipetted into the appropriate PCR tube by performing a visual inspection.
  3. Degradation or mishandling of the Positive Control itself

    The Positive Control may fail to amplify if it was not stored properly due to degradation of the target sequences in the COVID-19 Surveillance Positive Control. Ensure that all kit components are stored at -20 ºC when not in use. Also, ensure that the correct volume of Positive Control is transferred into the appropriate PCR reaction tube by performing visual inspections of the pipette tip.