Parvovirus PCR controls are essential to ensure the accuracy, sensitivity, and reliability of PCR assays used to detect parvovirus, particularly human parvovirus B19, which can cause a range of clinical conditions. Below is a detailed overview of the key controls used in Parvovirus PCR testing:

• Positive Control:
  • Purpose: Confirms that the PCR assay can effectively detect parvovirus DNA.
  • Content: Contains a known quantity of parvovirus B19 DNA or synthetic DNA sequences that correspond to the target regions of the parvovirus genome.
  • Procedure: This control is included in each PCR run to verify that the PCR reagents, primers, probes, and cycling conditions are functioning correctly. A positive result from this control confirms the assay's capability to detect parvovirus DNA, ensuring that the PCR process is working as intended.
• Negative Control:
  • Purpose: Detects contamination and prevents false positives.
  • Content: Includes all PCR reagents except for the target DNA, typically using nuclease-free water or a buffer solution.
  • Procedure: This control should show no amplification. If amplification occurs, it indicates possible contamination in the reagents, equipment, or laboratory environment. Such contamination necessitates investigation and potentially repeating the test to ensure accurate results.
• Internal Control:
  • Purpose: Ensures the integrity of the sample and the efficiency of the PCR process.
  • Content: Typically involves primers and probes targeting a non-parvovirus DNA sequence, such as a housekeeping gene from the host (e.g., human RNase P) or an exogenous control DNA/RNA spiked into the sample.
  • Procedure: This control is included in each sample to confirm that the sample processing, DNA extraction, and PCR amplification are functioning properly. If the internal control fails, it suggests issues with sample quality, DNA extraction, or the PCR conditions, which might affect the test's reliability.
• Extraction Control:
  • Purpose: Verifies the effectiveness of the DNA extraction process and checks for the presence of inhibitors.
  • Content: Typically involves a known quantity of non-target DNA (such as an exogenous control) or a small amount of parvovirus DNA.
  • Procedure: This control undergoes the same DNA extraction process as the test samples, ensuring that the extraction yields DNA of sufficient quality for PCR. Failure of the extraction control may indicate issues such as incomplete DNA extraction, degradation of DNA, or the presence of inhibitors that could interfere with PCR amplification.
• Blank Control:
  • Purpose: Ensures that the PCR setup environment is free from contamination.
  • Content: Consists of the complete PCR mix without any DNA template.
  • Procedure: This control should not produce any amplification. Detection of any signal in the blank control suggests contamination during the PCR setup, requiring decontamination of the workspace and potentially repeating the test to prevent false positives.
• Standard Curve Control (for Quantitative PCR):
  • Purpose: Assesses the quantitative accuracy and efficiency of the PCR assay.
  • Content: A series of known concentrations of parvovirus DNA.
  • Procedure: Used in quantitative PCR (qPCR) to generate a standard curve, which is then employed to quantify the amount of parvovirus DNA in the test samples. The accuracy of the viral load determination depends on the correct generation and interpretation of this standard curve.