CEN/TC 463 - Microbiology of the food chain

This document specifies the general principle and the technical protocol for the validation of alternative confirmation methods for microbiology in the food chain. This document compares the result of the alternative confirmation method against the confirmation procedure of a reference method or, if needed, a reference confirmation method (e.g. whole genome sequencing).
This document is applicable to the validation of alternative confirmation methods used for the analysis (detection or quantification) of isolated microorganisms in:
—          products intended for human consumption;
—          products intended for animal feeding;
—          environmental samples in the area of food and feed production, handling;
—          samples from the primary production stage.
Validated alternative confirmation methods can be used to replace (partly or completely) the confirmation procedure described in:
—          the reference method;
—          an alternative method validated in accordance with ISO 16140-2 only if one of the isolation agars specified in the validation study of the alternative confirmation method is used.
This document is also applicable to the validation of alternative typing methods, where the reference method can be, for example, a serological method (e.g. serotyping of Salmonella) or a molecular method (e.g. typing of Shiga toxin-producing E. coli).
This document is, in particular, applicable to bacteria and fungi. Some clauses can be applicable to other (micro)organisms, to be determined on a case-by-case basis.
Validation studies in accordance with this document are primarily intended to be performed by organizations or expert laboratories involved in method validation, but can also be used by a single laboratory, especially when performing in-house validation under certain conditions (see ISO 16140-4).

This document specifies requirements and gives guidance for the estimation and expression of measurement uncertainty (MU) associated with quantitative results in microbiology of the food chain.
It is applicable to the quantitative analysis of:
—          products intended for human consumption or the feeding of animals;
—          environmental samples in the area of food production and food handling;
—          samples at the stage of primary production.
The quantitative analysis is typically carried out by enumeration of microorganisms using a colony-count technique. This document is also generally applicable to other quantitative analyses, including:
—           most probable number (MPN) techniques;
—          instrumental methods, such as impediometry, adenosine triphosphate (ATP) and flow cytometry;
—          molecular methods, such as methods based on quantitative polymerase chain reaction (qPCR).
The uncertainty estimated by this document does not include systematic effects (bias).

This document specifies a method for detection of hepatitis A virus (HAV) and norovirus genogroups I (GI) and II (GII), from test samples of foodstuffs [(soft fruit, leaf, stem and bulb vegetables, bottled water, bivalve molluscan shellfish (BMS)] or surfaces using real-time RT-PCR.
This method is not validated for detection of the target viruses in other foodstuffs (including multi-component foodstuffs), or any other matrices, nor for the detection of other viruses in foodstuffs, surfaces or other matrices.

This document specifies protocols for conducting microbiological challenge tests for growth studies on vegetative and spore-forming bacteria in raw materials and intermediate or end products.
The use of this document can be extended to yeasts that do not form mycelium.

This document specifies requirements and gives guidelines for the organization of proficiency testing (PT) schemes for microbiological examinations of
a)   foods and beverages,
b)   feeding animals,
c)   environmental samples from food and feed production and handling, and
d)   primary production stages.
This document is also applicable to the microbiological examination of water where water is either used in food production or is regarded as a food in national legislation.
This document relates to the technical organization and implementation of PT schemes, as well as the statistical treatment of results of microbiological examinations.
This document is designed for use with ISO/IEC 17043 and ISO 13528, and deals only with areas where specific or additional details are necessary for PT schemes dealing with microbiological examinations for the areas specified in the first paragraph.

This document specifies horizontal methods for sampling techniques using contact plates, stick swabs, sponges and cloths on surfaces in the food chain environment in order to detect and enumerate culturable microorganisms such as pathogenic or non-pathogenic bacteria or yeasts and moulds.
NOTE       The term "environment" means any item in contact with the food product or likely to represent a contamination or recontamination source; for example, material, premises or operators.
This document does not apply to the validation of cleaning and disinfection procedures.
This document does not apply to sampling techniques for primary production samples, which are covered by ISO 13307. Sampling techniques for carcasses are covered by ISO 17604. Sampling techniques for analysis of noroviruses and hepatitis A viruses are covered by ISO 15216-1.
This document does not give advice on sampling frequency, the number of sampling points, or the need to rotate sampling points, as these are chosen on a case-by-case basis.

ISO 21872-1:2017 specifies a horizontal method for the detection of enteropathogenic Vibrio spp., which causes human illness in or via the intestinal tract. The species detectable by the methods specified include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
ISO 21872-1:2017 is applicable to the following:
-      products intended for human consumption and the feeding of animals;
-      environmental samples in the area of food production and food handling.

ISO 10272-2:2017 specifies a horizontal method for the enumeration of Campylobacter spp. It is applicable to
-      products intended for human consumption,
-      products intended for animal feeding,
-      environmental samples in the area of food and feed production, handling, and
-      samples from the primary production stage such as animal faeces, dust, and swabs.

ISO 19343:2017 specifies a high performance liquid chromatography (HPLC) method to analyse histamine in fish and fishery products (fish sauces, fish maturated by enzyme in brine, etc.) intended for human consumption.

ISO 10272-1:2017 specifies a horizontal method for the detection by enrichment or direct plating of Campylobacter spp. It is applicable to
-      products intended for human consumption,
-      products intended for animal feeding,
-      environmental samples in the area of food and feed production, handling, and
-      samples from the primary production stage such as animal faeces, dust, and swabs.

ISO 21528-1:2017 specifies a method, with enrichment, for the detection of Enterobacteriaceae. It is applicable to
-      products intended for human consumption and the feeding of animals, and
-      environmental samples in the area of primary production, food production and food handling.
This method is applicable
-      when the microorganisms sought are expected to need resuscitation by enrichment, and
-      when the number sought is expected to be below 100 per millilitre or per gram of test sample.
A limitation on the applicability of ISO 21528-1:2017 is imposed by the susceptibility of the method to a large degree of variability (see Clause 11).
NOTE       Enumeration can be carried out by calculation of the most probable number (MPN) after incubation in liquid medium. See Annex A.

ISO 21528-2:2017 specifies a method for the enumeration of Enterobacteriaceae. It is applicable to
-      products intended for human consumption and the feeding of animals, and
-      environmental samples in the area of primary production, food production and food handling.
This technique is intended to be used when the number of colonies sought is expected to be more than 100 per millilitre or per gram of the test sample.
The most probable number (MPN) technique, as included in ISO 21528‑1, is generally used when the number sought is expected to be below 100 per millilitre or per gram of test sample.

ISO 19020:2017 specifies a screening method for the detection of staphylococcal enterotoxins SEA, SEB, SECs, SED and SEE in foodstuffs. It consists of two main steps: a) extraction followed by a concentration based on dialysis principle; and b) an immunoenzymatic detection using commercially available detection kits.
ISO 19020:2017 is applicable to the screening of staphylococcal enterotoxins SEA to SEE in products intended for human consumption.
Other staphylococcal enterotoxins such as types SEG, SEH, SEI, SER, SES and SET can also cause illness. Due to the lack of commercially available detection kits, ISO 19020:2017 is applicable only to types SEA to SEE, but may apply to other types of toxins, subject to validation of the method.

ISO 11290-1:2017 specifies a horizontal method for
-      the detection of L. monocytogenes, and
-      the detection of Listeria spp. (including L. monocytogenes).
ISO 11290-1:2017 is applicable to
-      products intended for human consumption and for the feeding of animals, and
-      environmental samples in the area of food production and food handling.

ISO 11290-2:2017 specifies a horizontal method for
-      the enumeration of L. monocytogenes, and
-      the enumeration of Listeria spp. (including L. monocytogenes).
ISO 11290-2:2017 is applicable to
-      products intended for human consumption and for the feeding of animals, and
-      environmental samples in the area of food production and food handling.

ISO 22964:2017 specifies a horizontal method for the detection of Cronobacter spp.
Subject to the limitations discussed in the introduction, this document is applicable to
-      food products and ingredients intended for human consumption and the feeding of animals, and
-      environmental samples in the area of food production and food handling.

ISO 10273:2017 specifies a horizontal method for the detection of Y. enterocolitica associated with human disease. It is applicable to
-      products intended for human consumption and the feeding of animals, and
-      environmental samples in the area of food production and food handling.

This standard describes the detection of E.coli O157 (Reference document EN ISO 16654)

ISO 6887-4:2017 specifies rules for the preparation of samples and dilutions for the microbiological examination of specific food products not covered in other parts of ISO 6887, which deal with more general categories. This document covers a wide range of miscellaneous products, but does not include new products brought on to the market after publication.
ISO 6887‑1 defines the general rules for the preparation of the initial suspension and dilutions for microbiological examination.
ISO 6887-4:2017 excludes preparation of samples for both enumeration and detection test methods when preparation details are specified in the relevant International Standards.
ISO 6887-4:2017 is applicable to the following products:
-      acidic (low pH) products;
-      hard and dry products;
-      dehydrated, freeze-dried and other low aw products (including those with inhibitory properties);
-      flours, whole cereal grains, cereal by-products;
-      animal feed, cattle cake, kibbles and pet chews;
-      gelatine (powdered and leaf);
-      margarines, spreads and non-dairy products with added water;
-      eggs and egg products;
-      bakery goods, pastries and cakes;
-      fresh fruit and vegetables;
-      fermented products and other products containing viable microorganisms;
-      alcoholic and non-alcoholic beverages;
-      alternative protein products.

ISO 6887-2:2017 specifies rules for the preparation of meat and meat product samples and their suspension for microbiological examination when the samples require different preparation from the methods described in ISO 6887‑1. ISO 6887‑1 defines the general rules for the preparation of the initial suspension and dilutions for microbiological examination.
ISO 6887-2:2017 excludes preparation of samples for both enumeration and detection test methods where preparation details are specified in the relevant International Standards.
ISO 6887-2:2017 is applicable to the following fresh, raw and processed meats, poultry and game and their products:
-      refrigerated or frozen;
-      cured or fermented;
-      minced or comminuted;
-      meat preparations;
-      mechanically separated meat;
-      cooked meats;
-      dried and smoked meats at various degrees of dehydration;
-      concentrated meat extracts;
-      excision and swab samples from carcasses.
ISO 6887-2:2017 excludes the sampling of carcasses (see ISO 17604) and preparation of samples from the primary production stage (see ISO 6887‑6).

ISO 6887-3:2017 specifies rules for the preparation of fish and fishery product samples and their suspension for microbiological examination when the samples require a different preparation from the methods described in ISO 6887‑1. ISO 6887‑1 defines the general rules for the preparation of the initial suspension and dilutions for microbiological examination.
ISO 6887-3:2017 includes special procedures for sampling raw molluscs, tunicates and echinoderms from primary production areas.
NOTE 1       Sampling of raw molluscs, tunicates and echinoderms from primary production areas is included in this document, rather than ISO 13307, which specifies rules for sampling from the terrestrial primary production stage.
ISO 6887-3:2017 excludes preparation of samples for both enumeration and detection test methods where preparation details are specified in the relevant International Standards (e.g. ISO/TS 15216‑1 and ISO/TS 15216‑2 for determination of hepatitis A virus and norovirus in food using real-time RT-PCR).
ISO 6887-3:2017 is intended to be used in conjunction with ISO 6887‑1. It is applicable to the following raw, processed or frozen fish and shellfish and their products (see Annex A for classification of major taxa):
a)    Raw fishery products, molluscs, tunicates and echinoderms including:
-      whole fish or fillets, with or without skin and heads, and gutted;
-      crustaceans, whole or shelled;
-      cephalopods;
-      bivalve molluscs;
-      gastropods;
-      tunicates and echinoderms.
b)    Processed products including:
-      smoked fish, whole or prepared fillets, with or without skin;
-      cooked or partially cooked, whole or shelled crustaceans, molluscs, tunicates and echinoderms;
-      cooked or partially cooked fish and fish-based multi-component products.
c)    Raw or cooked frozen fish, crustaceans, molluscs and others, in blocks or otherwise, including:
-      fish, fish fillets and pieces;
-      whole and shelled crustacean (e.g. flaked crab, prawns), molluscs, tunicates and echinoderms.
NOTE 2       The purpose of examinations performed on these samples can be either hygiene testing or quality control. However, the sampling techniques described in this document relate mainly to hygiene testing (on muscle tissues).

ISO 6887-1:2017 defines general rules for the aerobic preparation of the initial suspension and of dilutions for microbiological examinations of products intended for human or animal consumption.
ISO 6887-1:2017 is applicable to the general case and other parts apply to specific groups of products as mentioned in the foreword. Some aspects might also be applicable to molecular methods where matrices can be associated with inhibition of the PCR steps and consequently affect the test result.
ISO 6887-1:2017 excludes preparation of samples for both enumeration and detection test methods where preparation instructions are detailed in specific International Standards.

ISO 15216-1:2017 specifies a method for the quantification of levels of HAV and norovirus genogroup I (GI) and II (GII) RNA, from test samples of foodstuffs (soft fruit, leaf, stem and bulb vegetables, bottled water, BMS) or food surfaces. Following liberation of viruses from the test sample, viral RNA is then extracted by lysis with guanidine thiocyanate and adsorption on silica. Target sequences within the viral RNA are amplified and detected by real-time RT-PCR.
This method is not validated for detection of the target viruses in other foodstuffs (including multi-component foodstuffs), or any other matrices, nor for the detection of other viruses in foodstuffs, food surfaces or other matrices.

ISO 6579-1:2017 specifies a horizontal method for the detection of Salmonella. It is applicable to the following:
-      products intended for human consumption and the feeding of animals;
-      environmental samples in the area of food production and food handling;
-      samples from the primary production stage such as animal faeces, dust, and swabs.
With this horizontal method, most of the Salmonella serovars are intended to be detected. For the detection of some specific serovars, additional culture steps may be needed. For Salmonella Typhi and Salmonella Paratyphi, the procedure is described in Annex D.
The selective enrichment medium modified semi-solid Rappaport-Vassiliadis (MSRV) agar is intended for the detection of motile Salmonella and is not appropriate for the detection of non-motile Salmonella strains.

ISO 18465:2017 describes the quantitative analysis of the emetic toxin cereulide using high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UHPLC) connected to a tandem mass spectrometer (LC-MS/MS).
ISO 18465:2017 is applicable to the analysis of the toxin in products intended for human consumption.

ISO 16140-1:2016 defines general terms and definitions relating to method validation of microbiology in the food chain.
It is applicable to the validation of methods for the analysis (detection or quantification) of microorganisms in
-      products intended for human consumption,
-      products intended for animal feeding,
-      environmental samples in the area of food and feed production, handling, and
-      samples from the primary production stage.

ISO 16140-2:2016 specifies the general principle and the technical protocol for the validation of alternative, mostly proprietary, methods for microbiology in the food chain. Validation studies according to ISO 16140-2:2016 are intended to be performed by organizations involved in method validation.
It is applicable to the validation of methods for the analysis (detection or quantification) of microorganisms in
-      products intended for human consumption,
-      products intended for animal feeding,
-      environmental samples in the area of food and feed production, handling, and
-      samples from the primary production stage.
It is in particular applicable to bacteria and fungi. Some clauses of ISO 16140-2:2016 could be applicable to other (micro) organisms or their metabolites on a case-by-case-basis. In the future, guidance for other organisms (e.g. viruses and parasites) will be included in ISO 16140:2016 (all parts).

ISO 17468:2016 gives technical requirements and guidance on the establishment or revision of standardized reference methods for the analysis (detection or quantification) of microorganisms in
-      products intended for human consumption and for the feeding of animals,
-      environmental samples in the area of food/feed production and food/feed handling, and
-      samples from the primary production stage.
It defines the technical stage (or early stage) of the establishment of a new standardized reference method or of the revision of an existing standardized reference method. It includes, in particular, requirements and guidance on the validation of the selected method.
It is intended to be implemented in particular by ISO/TC 34/SC 9 and its corresponding structure at CEN level, CEN/TC 275, Food analysis - Horizontal methods, Working Group 6, Microbiology of the food chain.

ISO 18744:2016 specifies a method that is applicable for the detection and enumeration of Cryptosporidium oocysts and Giardia cysts on or in food products that are described herein as fresh leafy green vegetables and berry fruits. With suitable controls, it may also be applicable for the examination of other fresh produce.
The microscopy descriptions are for Cryptosporidium spp. oocysts and Giardia duodenalis cysts of size ranges which include those species (Cryptosporidium) or assemblages (Giardia) known to be pathogenic to humans.
This method does not include any molecular analysis and therefore is not suitable for the determination of the species or genotypes/assemblages of Cryptosporidium oocysts and Giardia cysts. The method will detect all species and genotypes/assemblages that are known to be pathogenic for humans and also others that are not. For further identification, molecular typing assays are required. However, these cannot be reliably performed if process positive controls have been spiked into the samples, as the result of molecular typing assays will be obfuscated.
This method does not allow the determination of viability or infectivity of any Cryptosporidium oocysts and Giardia cysts which may be present.

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 18743:2015 specifies a method of detection of Trichinella spp. muscle stage larvae in meat of individual animal carcasses intended for human consumption. It is applicable to the examination of meat from domestic and sylvatic animal species, which can be infected by nematodes of the genus Trichinella.
This method does not allow the identification of the species or genotype of detected parasites; species or genotype identification can be carried out by molecular methods.
The method described in this International Standard is intended to be used in conjunction with the guidelines in the OIE Manual of Diagnostic Tests and Vaccines and by the International Commission on Trichinellosis (ICT) for Trichinella testing and the inspection of carcasses intended for human consumption, unless it has been demonstrated by other means that the animal was not at risk for exposure to Trichinella.
The artificial digestion/magnetic stirrer method is considered to be the standard method because it has proven to give the most reliable results in validation studies.
NOTE          Provided equivalence with the method described within this International Standard can be documented, alternative methods can be used for analysis.

ISO 17604:2015 specifies sampling methods for the detection and enumeration of microorganisms on the surface of carcasses or parts of carcasses of slaughtered meat animals. The microbiological sampling can be carried out as part of
-      process hygiene control (to validate and or verify process control, e.g. total counts and Enterobacteriaceae) in slaughter establishments for large mammals, poultry, and game,
-      risk-based assurance systems for product safety, and
-      monitoring or surveillance programmes for the prevalence and/or numbers of pathogenic microorganisms.
This International Standard includes the use of excision and swabbing techniques depending on the reason for sample collection. It also includes the use of carcass rinsing for the examination of carcasses of poultry and some small animals. Annex A shows sampling sites on the carcasses of various animal species.

ISO/TS 17728:2015 applies to the collection of samples before submission to the laboratory for microbiological examination. It provides general instructions and specific requirements for obtaining samples and for transport to the laboratory.
Sampling plans are not included in the scope of this Technical Specification.

ISO 16649-3:2015 specifies a horizontal method for the detection and enumeration of β-glucuronidase positive Escherichia coli, by means of the liquid-medium culture technique and calculation of the most probable number (MPN) after incubation at (37 ± 1) °C, then at (44 ± 1) °C. This part of ISO 16649 is applicable to the following:
-      products intended for human consumption and the feeding of animals;
-      environmental samples in the area of food production and food handling.
The method is suitable for the enumeration of cells of E. coli that might have been subjected to stress arising from dehydration, freezing, and exposure to a saline (such as marine) environment or damage by disinfectants such as chlorine-containing products.
A limitation of the applicability of this part of ISO 16649 is imposed by the susceptibility of the method to a large degree of variability. The method is intended to be applied and the results interpreted in the light of the information given in Clause 11.
This method has not been fully evaluated for all matrices (e.g. for milk and milk products). ISO 7251 is intended to be used for milk and milk products.

ISO/TR 6579-3:2014 gives guidance on the procedure for serotyping Salmonella serovars and is applicable to the serotyping of pure cultures of Salmonella spp., independent of the source from which they are isolated.

ISO 11133:2014 defines terms related to quality assurance of culture media and specifies the requirements for the preparation of culture media intended for the microbiological analysis of food, animal feed, and samples from the food or feed production environment as well as all kinds of water intended for consumption or used in food production. These requirements are applicable to all categories of culture media prepared for use in laboratories performing microbiological analyses.
ISO 11133:2014 also sets criteria and describes methods for the performance testing of culture media. It applies to producers such as:
commercial bodies producing and/or distributing ready-to-use or semi-finished reconstituted or dehydrated media;
non-commercial bodies supplying media to third parties;
microbiological laboratories preparing culture media for their own use.

ISO corrigendum

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 4833-2:2013 specifies a horizontal method for enumeration of microorganisms that are able to grow and form colonies on the surface of a solid medium after aerobic incubation at 30 °C. The method is applicable to:
a)    products intended for human consumption or for animal feed;
b)    environmental samples in the area of food and feed production and food handling.
ISO 4833-2:2013 is applicable to:
1)    products containing heat-sensitive organisms that are likely to form a significant proportion of the total flora (e.g. psychrotrophic organisms in chilled and frozen foods, dried foods, other foods that may contain heat-sensitive organisms);
2)    products containing obligately aerobic bacteria that are likely to form a significant proportion of the total flora (e.g. Pseudomonas spp.);
3)    products that contain small particles that can prove difficult to distinguish from colonies in a pour plate;
4)    products whose intense colour prevents the recognition of colonies in a pour plate;
5)    products for which distinction between different types of colony is required as part of the assessment of food quality.
In addition to the manual spread plating technique, an annex to ISO 4833-2:2013 also specifies the use of a spiral plater, a rapid method of performing surface colony counts.
The applicability of ISO 4833-2:2013 to the examination of certain fermented food and animal feeds is limited and other media or incubation conditions can be more appropriate. However, this method can be applied to such products even though it is possible that the predominant microorganisms in these products are not detected effectively.
For some matrices, the method described in ISO 4833-2:2013 can give different results to those obtained using the method described in ISO 4833‑1.

ISO 4833-1:2013 specifies a horizontal method for enumeration of microorganisms that are able to grow and form colonies in a solid medium after aerobic incubation at 30 °C. The method is applicable to: a) products intended for human consumption and for animal feed; b) environmental samples in the area of food and feed production and handling.
ISO 4833-1:2013 is applicable to: 1) products that require a reliable count when a low limit of detection is specified (below 102/g or 102/ml for liquid samples or below 103/g for solid samples); 2) products expected to contain spreading colonies that obscure colonies of other organisms, e.g. milk and milk products likely to contain spreading Bacillus spp.
The applicability of ISO 4833-1:2013 to the examination of certain fermented food and animal feeds is limited and other media or incubation conditions can be more appropriate. However, this method can be applied to such products even though it is possible that the predominant microorganisms in those products are not detected effectively.
For some matrices, the method specified in ISO 4833-1:2013 can give different results to those obtained using the method specified in ISO 4833‑2.

2012-12-04 GVN: Draft for // vote received at ISO/CS (see notification in dataservice on 2012-12-01)

ISO 13307:2013 specifies sampling techniques within the primary food-animal production stage, for detection or enumeration of viable microorganisms with particular reference to food-borne pathogens.
ISO 13307:2013 is not intended for use in diagnosis of animal disease.

ISO 6887-6:2013 specifies rules for the preparation of samples taken at all stages from the farm to the slaughterhouse and their suspension for microbiological examination when the samples require different preparation from the methods described in ISO 6887-1.
ISO 6887-6:2013 excludes the preparation of samples for both enumeration and detection test methods where preparation details are specified in the relevant International Standards.
ISO 6887-6:2013 is applicable to various samples taken from the hatchery, the farm, from the vehicle or the animals during transportation, or from animals or their carcasses in the slaughterhouse, to indicate the microbiological status of the animals in relation to zoonotic agents. ISO 6887-6:2013 does not apply to samples taken to assess the hygiene of meat.

ISO/TR 13136:2012 describes the identification of Shiga toxin-producing Escherichia coli (STEC) by means of the detection of the following genes: a) the major virulence genes of STEC, stx and eae; b) the genes associated with the serogroups O157, O111, O26, O103, and O145.
In any case, when one or both of the stx genes is/are detected, the isolation of the strain is attempted.
The isolation of STEC from samples positive for the presence of the genes specifying the serogroups in the scope of this method can be facilitated by using serogroup-specific enrichment techniques (e.g. immunomagnetic separation, IMS).
The protocol uses real-time PCR as the reference technology for detection of the virulence and serogroup-associated genes.
ISO/TR 13136:2012 is applicable to: 1) products intended for human consumption and the feeding of animals; 2) environmental samples in the area of food production and food handling; 3) environmental samples in the area of primary production.

ISO/TS 6579-2 gives a method for the enumeration of Salmonella spp. present in: products intended for human consumption and for the feeding of animals; environmental samples in the area of food production and food handling; animal faeces; and environmental samples from the primary production stage by calculation of the most probable number (MPN).
The method is based on miniaturization of the dilution, pre-enrichment and selective enrichment steps. The selective enrichment medium, modified semi-solid Rappaport?Vassiliadis (MSRV), is intended for the detection of motile salmonellae and is not appropriate for the detection of non-motile salmonellae.
It is possible that the method is less appropriate to enumerate Salmonella ser. Typhi and Salmonella ser. Paratyphi.
The method is not appropriate for the enumeration of Salmonella spp. in (very) low contaminated samples (<1 cfu/g).

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.