CEN/TC 463/WG 1 - General requirements relating to PCR methods

This document specifies the general requirements for the in vitro amplification of nucleic acid sequences (DNA or RNA).
This document is applicable to the testing for microorganisms and viruses from the food chain using the polymerase chain reaction (PCR). This document, or parts of it, is applicable to other fields of PCR diagnostics based on a case-by-case evaluation.
The minimum requirements laid down in this document are intended to ensure that comparable and reproducible results are obtained in different laboratories.
This document has been established for microorganisms from the food chain and is applicable to:
—     products intended for human consumption;
—     products for feeding animals;
—     environmental samples in the area of food and feed production and handling;
—     samples from the primary production stage.

This document specifies requirements for the installation, maintenance, temperature calibration and temperature performance testing of standard thermal cyclers and real-time thermal cyclers. It is applicable to the detection of microorganisms as well as any other applications in the food chain using polymerase chain reaction (PCR)-based methods.
This document has been established for food testing, but is also applicable to other domains using thermal cyclers (e.g. environmental, human health, animal health, forensic testing).

ISO/TS 18867:2015 specifies two horizontal methods for detection of the pathogenic bioserotypes of Y. enterocolitica and one for detection of Y. pseudotuberculosis by using real-time PCR-based methods. The described methods allow for the detection of the two pathogens in enrichments and allow the isolation of colonies. Y. pestis, the causative agent of bubonic and pneumonic plague harbours a variant of the ail gene as well and will be detected by the same primer/probe set as Y. pseudotuberculosis. However, Y. pestis is normally not associated with food. This Technical Specification is applicable to products for human consumption, animal feeding stuffs, and environmental samples.

ISO/TS 17919:2013 specifies a horizontal method for the molecular detection of clostridia carrying botulinum neurotoxin A, B, E, and F genes by a PCR method. This method detects the genes and not the toxins, therefore a positive result does not necessarily mean the presence of these toxins in the sample investigated. ISO/TS 17919:2013 is applicable to products for human consumption, animal feed, and environmental samples.
The PCR assays for detection of genetic sequences encoding specific toxin types are described in annexes.

ISO 22118:2010 specifies minimum requirements of performance characteristics for the detection of nucleic acid sequences (DNA or RNA) by molecular methods. ISO 22118:2010 applies to the detection of food-borne pathogens in foodstuffs and isolates obtained from them using molecular detection methods based on the polymerase chain reaction (PCR).
ISO 22118:2010 is also applicable, for example, to the detection of food-borne pathogens in environmental samples and in animal feeding stuffs.

ISO 22119:2010 defines terms for the detection of food-borne pathogens in foodstuffs, and isolates obtained from them, using the polymerase chain reaction (PCR). ISO 22119:2010 specifies requirements for the amplification and detection of nucleic acid sequences (DNA or RNA after reverse transcription) by real-time PCR.
The minimum requirements laid down in ISO 22119:2010 provide the basis for comparable and reproducible results within individual and between different laboratories.
ISO 22119:2010 is also applicable, for example, to the detection of food-borne pathogens in environmental samples and in animal feeding stuffs.

ISO 20837:2006 provides criteria and examples for sample preparation in order to obtain PCR-compatible samples or nucleic acids of suitable quality and quantity for PCR.
It provides a description of the general principles involved. References to standards concerning the enrichment of microorganisms are given in Annex A, and a detailed method for DNA extraction is given in Annex B.
ISO 20837:2006 has been established for food matrices, but could also be applied to feed and agricultural/environmental matrices with some adaptations, if necessary.

ISO 20838:2006 provides the overall framework for qualitative methods for the detection of food-borne pathogens using the polymerase chain reaction (PCR).
It covers the general requirements for the specific amplification of target nucleic acid sequences and the detection and confirmation of the identity of the amplified nucleic acid sequence.
Guidelines, minimum requirements and performance characteristics described in ISO 20838:2006 are intended to ensure that comparable and reproducible results are obtained in different laboratories.
ISO 20838:2006 has been established for food-borne pathogens in or isolated from food and feed matrices, but can also be applied to other matrices, for example environmental samples, or to the detection of other microorganisms under investigation.

ISO 22174:2004 gives the general requirements for the in vitro amplification of nucleic acid sequences (DNA or RNA). It is applicable to the testing of foodstuffs and isolates obtained from foodstuffs for food-borne pathogens using the polymerase chain reaction (PCR)
The minimum requirements laid down in ISO 22174:2004 are intended to ensure that comparable and reproducible results are obtained in different laboratories.
ISO 22174:2004 has been established for food-borne pathogens in or isolated from food and feed matrices, but is also applicable to other matrices (e.g. environmental samples) and for the detection of non-pathogenic microorganisms.

ISO/TS 20836:2005 provides basic requirements for the installation, performance and maintenance of thermal cyclers. Although thermal cyclers are robust technical equipment, they do require regular maintenance. Their cooling/heating elements have a limited lifetime. Proper functioning of the cooling/heating element depends both on the quality of the cooling/heating devices and proper use and care.
In addition to outlining the requirement for a defined maintenance programme, procedures are described for the determination of thermal cycler performance by biochemical or physical methods.