Table 1.

Analytical controls for (RT) real‐time PCR‐based detection of viral hazards in food matrices

Process controls

Processing Positive Control (PPC): A negative sample spiked with sufficient viral target and processed throughout the entire protocol. A positive signal should be obtained indicating that the entire process was correctly performed

Processing Negative Control (PNC): A negative sample spiked with sufficient amount of nontarget or water and processed throughout the entire protocol. A negative signal should be obtained, indicating the lack of contamination throughout the entire process. For example, the inclusion of encapsidated RNA (or DNA) or bacteriophages

Environmental Control: A tube containing the master mixture or water left open in the PCR set‐up room to detect possible contaminating nucleic acids in the environment

Amplification controls

Positive PCR control: A viral template known to contain the target sequence. Positive amplification indicates that amplification was performed correctly. It could be used a natural virus or chimerical nucleic acids

Negative PCR control (or No Template Control ‐NTC‐ or Reagent Control or Blank): Including all reagents used in the amplification except the template nucleic acids. Usually, water is added instead of the template. A negative signal indicates the absence of specific contamination in the amplification assay

External Amplification control (EAC): An aliquot of a solution of control DNA, containing a defined quantity or copy number, added to an aliquot of the nucleic acid of the extracted sample and analysed in a separate reaction tube. A positive signal indicates that the sample nucleic acid extract did not contain any inhibitory substances

Internal Amplification Control (IAC): Chimerical nontarget nucleic acid added to the master mix to be co‐amplified with the same primer set as the viral target but with an amplicon size visually distinguishable or different internal sequence region from the target amplicon. The amplification of the IAC both in the presence and in the absence of the target indicates that the amplification conditions are adequate

Adapted from Rodríguez‐Lázaro et al. (2007), Pintó & Bosch (2008), Bosch et al. (2011) and D'Agostino et al. (2011).

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