Norovirus PCR controls are essential for ensuring the accuracy, sensitivity, and reliability of PCR assays designed to detect norovirus. Here's a technical overview of the key controls used in Norovirus PCR testing:

• Positive Control:
  • Purpose: Validates that the PCR assay can successfully detect norovirus RNA.
  • Content: Contains a known quantity of norovirus RNA (typically from a well-characterized norovirus strain) or synthetic RNA mimicking the target norovirus sequences.
  • Procedure: This control is included in every PCR run to verify that the PCR reagents, primers, probes, and overall assay conditions are functioning correctly. A positive result from this control confirms that the PCR process is working as intended.
• Negative Control:
  • Purpose: Detects contamination and ensures that no false positives occur.
  • Content: Typically includes all PCR reagents except the target RNA, such as water or a buffer solution.
  • Procedure: This control should yield no amplification. If amplification occurs, it indicates potential contamination in the reagents or the laboratory environment, which would necessitate retesting and possible decontamination.
• Internal Control:
  • Purpose: Confirms the integrity of the sample and the overall PCR process.
  • Content: Usually consists of a non-norovirus RNA target, such as an endogenous human gene (e.g., RNase P) or an exogenous RNA control added to the sample.
  • Procedure: This control is included in each sample to verify that the sample was correctly processed and that the PCR amplification process is functioning properly. A failed internal control may indicate issues with sample quality, RNA extraction, or the PCR reaction itself.
• Extraction Control:
  • Purpose: Ensures that the RNA extraction process was effective and free from inhibitors.
  • Content: May include a known concentration of a non-target RNA (like a spiked-in exogenous control) or a portion of norovirus RNA.
  • Procedure: This control undergoes the same RNA extraction process as the test samples, ensuring that the extraction was successful and that the RNA is of sufficient quality for PCR. Failure to detect this control may indicate problems with the extraction process, such as RNA degradation or the presence of inhibitors.
• Blank Control:
  • Purpose: Ensures that there is no contamination during the PCR setup.
  • Content: Comprises the complete PCR mixture without any nucleic acid template.
  • Procedure: Similar to the negative control, the blank control should not produce any amplification. Any positive signal suggests contamination during the PCR setup, necessitating decontamination and retesting.
• Standard Curve Control (for Quantitative PCR):
  • Purpose: Assesses the accuracy and efficiency of the quantitative PCR assay.
  • Content: A series of known concentrations of norovirus RNA.
  • Procedure: Used in qPCR to generate a standard curve, which is then used to determine the viral load in test samples. Accurate quantification relies on the correct generation and interpretation of this standard curve.