Rotavirus PCR controls are essential for ensuring the accuracy, reliability, and validity of PCR assays used to detect rotavirus RNA. Below is a detailed overview of the key controls used in Rotavirus PCR testing:

• Positive Control:
  • Purpose: Confirms that the PCR assay can effectively detect rotavirus RNA.
  • Content: Contains a known concentration of rotavirus RNA or synthetic RNA sequences that mimic the target regions of the rotavirus 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 from this control confirms the assay's capability to detect rotavirus RNA, ensuring the PCR process is working as intended.
• Negative Control:
  • Purpose: Detects contamination and prevents false positives.
  • Content: Includes all PCR reagents except for 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, equipment, or laboratory environment. In such cases, an investigation and possibly a repeat of the test are necessary to ensure accurate results.
• Internal Control:
  • Purpose: Ensures the integrity of the sample and the efficiency of the PCR process.
  • Content: Often includes primers and probes targeting a non-rotavirus RNA sequence, such as a housekeeping gene from the host (e.g., human RNase P) or an exogenous RNA control spiked into the sample.
  • Procedure: This control is included in each sample to confirm that sample processing, RNA extraction, and PCR amplification are functioning properly. A failure in this control suggests issues with sample quality, RNA extraction, or PCR conditions.
• Extraction Control:
  • Purpose: Confirms the effectiveness of the RNA extraction process and checks for the presence of inhibitors.
  • Content: Typically involves a known quantity of non-target RNA (such as an exogenous control) or a small amount of rotavirus RNA.
  • Procedure: This control undergoes the same RNA extraction process as the test samples, ensuring that the extraction yields RNA of sufficient quality for PCR. If the extraction control fails, it may indicate problems with the extraction procedure, such as incomplete lysis, RNA degradation, or the presence of inhibitors that could interfere with the PCR.
• Blank Control:
  • Purpose: Ensures that the PCR setup environment is free from contamination.
  • Content: Comprises the complete PCR mixture without any RNA template.
  • Procedure: This control should not produce any amplification. Any signal detected in the blank control suggests contamination during the PCR setup, requiring decontamination of the workspace and potentially repeating the test.
• Standard Curve Control (for Quantitative PCR):
  • Purpose: Assesses the quantitative accuracy and efficiency of the PCR assay.
  • Content: A series of known concentrations of rotavirus RNA.
  • Procedure: Used in quantitative PCR (qPCR) to generate a standard curve, which is then used to quantify the amount of rotavirus RNA in the test samples. Accurate quantification depends on the proper generation and interpretation of this standard curve.