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

• Positive Control:
  • Purpose: Confirms that the PCR assay can successfully detect Zika virus RNA.
  • Content: Contains a known quantity of Zika virus RNA or synthetic RNA sequences that match the target regions of the Zika virus genome.
  • Procedure: This control is included in each PCR run to verify that the PCR reagents, primers, probes, and thermal cycling conditions are functioning correctly. A positive result indicates that the assay is capable of detecting Zika virus RNA.
• Negative Control:
  • Purpose: Detects contamination and prevents false positives.
  • Content: Includes all PCR reagents except the RNA template, typically using nuclease-free water or a buffer solution.
  • Procedure: This control should yield no amplification. If amplification occurs, it indicates contamination in the reagents or the laboratory environment, necessitating an investigation and retesting.
• Internal Control:
  • Purpose: Ensures the integrity of the sample and the overall PCR process.
  • Content: Often includes primers and probes targeting a non-Zika RNA sequence, such as a human housekeeping gene (e.g., RNase P) or an exogenous control RNA added to the sample.
  • Procedure: This control is included in each sample to confirm that the RNA extraction and PCR amplification processes are functioning properly. A failed internal control suggests issues with the sample quality, RNA extraction, or the PCR assay itself.
• Extraction Control:
  • Purpose: Confirms the efficiency of the RNA extraction process and the absence of inhibitors.
  • Content: May include a known quantity of a non-target RNA (e.g., an exogenous RNA spike-in) or a portion of Zika RNA.
  • Procedure: This control undergoes the same RNA extraction process as the test samples to ensure that the extraction is successful and that the RNA is of sufficient quality for PCR. Failure to detect this control can indicate problems with the extraction process, such as degraded RNA 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 RNA template.
  • Procedure: This control should not show any amplification. Any signal detected in the blank control suggests contamination during the PCR setup, requiring decontamination of the workspace and retesting.
• Standard Curve Control (for Quantitative PCR):
  • Purpose: Evaluates the quantitative accuracy and efficiency of the PCR assay.
  • Content: A series of known concentrations of Zika virus RNA.
  • Procedure: Used in quantitative PCR (qPCR) to create a standard curve, which is then used to determine the viral load in the test samples. Proper calibration of the standard curve is essential for accurate quantification.