1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationMicrobiology of food and animal feed Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens Horizontal method for the detection of Shiga
2、 toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroupsPD CEN ISO/TS 13136:2012National forewordThis Published Document is the UK implementation of CEN ISO/TS 13136:2012.The UK participation in its preparation was entrusted to Technical Committee A
3、W/9, Microbiology.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2012.Publi
4、shed by BSI Standards Limited 2012 ISBN 978 0 580 67595 9 ICS 07.100.30Compliance with a British Standard cannot confer immunity from legal obligations.This Published Document was published under the authority of the Standards Policy and Strategy Committee on 30 November 2012.Amendments issued since
5、 publicationDate Text affectedPUBLISHED DOCUMENTPD CEN ISO/TS 13136:2012TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN ISO/TS 13136 November 2012 ICS 07.100.30 English Version Microbiology of food and animal feed - Real-time polymerase chain reaction (PCR)-based method f
6、or the detection of food-borne pathogens - Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups (ISO/TS 13136:2012) Microbiologie des aliments - Mthode base sur la raction de polymrisation en chane (PC
7、R) en temps rel pour la dtection des micro-organismes pathognes dans les aliments - Mthode horizontale pour la dtection des Escherichia coli producteurs de Shigatoxines (STEC) et la dtermination des srogroupes O157, O111, O26, O103 et O145 (ISO/TS 13136:2012) Mikrobiologie von Lebensmitteln und Futt
8、ermitteln - Real-time-Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln - Horizontales Verfahren fr den Nachweis von Shiga-Toxin bildenden Escherichia coli (STEC) der Serogruppen O157, O111, O26, O103 und O145 (ISO/TS 13136:2012) This Technical Specificatio
9、n (CEN/TS) was approved by CEN on 16 July 2012 for provisional application. The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their comments, particularly on the question whether the CEN/TS can be converted into
10、a European Standard. CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS) until
11、the final decision about the possible conversion of the CEN/TS into an EN is reached. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Icel
12、and, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre
13、: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN ISO/TS 13136:2012: EPD CEN ISO/TS 13136:2012CEN ISO/TS 13136:2012 (E) 2 Contents Page Foreword 3PD CEN ISO/TS 13136:2012CEN ISO/TS 13136:2012
14、 (E) 3 Foreword This document (CEN ISO/TS 13136:2012) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is held by DIN, in collaboration with Technical Committee ISO/TC 34 “Food products“. Attention is drawn to the possibility that some
15、 of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to annou
16、nce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Rom
17、ania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN ISO/TS 13136:2012ISO/TS 13136:2012(E) ISO 2012 All rights reserved iiiContents PageIntroduction v 1 Scope 1 2 Normative references . 1 3 Terms and definitions . 2 4 Principle . 24.1 General . 24.2 Microbial e
18、nrichment 24.3 Nucleic acid extraction 34.4 Target genes . 34.5 Detection 34.6 Isolation . 35 Diluents, culture media and reagents 35.1 Culture media . 45.2 Reagents for nucleic acid extraction . 55.3 Reagents for PCR 56 Equipment . 57 Sampling 68 Preparation of test sample 69 Procedure 69.1 Test po
19、rtion and initial suspension 69.2 Enrichment 79.3 Nucleic acid extraction 79.4 PCR amplification (for real-time PCR) . 79.5 Strain isolation . 8 10 Expression of results . 8 11 Performance data 8 Annex A (normative) Flow diagram of the screening procedure .12 Annex B (normative) Flow diagram of the
20、isolation and confirmation procedure .13 Annex C (informative) Identification of Shiga toxin-producing Escherichia coli (STEC) by multiplex PCR amplification of virulence genes and detection of PCR products by agarose gel electrophoresis .14Annex D (informative) Internal amplification control 18 Ann
21、ex E (informative) Primers and probes for the PCR assays 19 Annex F (normative) Isolation of STEC strains 21 Bibliography .22PD CEN ISO/TS 13136:2012ISO/TS 13136:2012(E)IntroductionShiga toxin-producing Escherichia coli (STEC) are pathogenic E. coli, which can cause diarrhoea as well as more severe
22、diseases in humans such as haemorrhagic colitis and haemolytic uremic syndrome (HUS). Although STEC may belong to a large number of serogroups, those that have been firmly associated with the most severe forms of the disease, in particular HUS, belong to O157, O26, O111, O103, and O145 (Reference 1)
23、.The following nomenclature has been adopted in this Technical Specification: stx: Shiga toxin genes (synonymous with vtx); Stx: Shiga toxin (synonymous with Vtx: Verocytotoxin); STEC: Shiga toxin-producing Escherichia coli (synonymous with VTEC: Verocytotoxin-producing Escherichia coli).The Interna
24、tional Organization for Standardization (ISO) draws attention to the fact that it is claimed that compliance with this document may involve the use of patents.ISO takes no position concerning the evidence, validity and scope of this patent right.The holder of this patent right has assured ISO that h
25、e/she is willing to negotiate licences either free of charge or under reasonable and non-discriminatory terms and conditions with applicants throughout the world. In this respect, the statement of the holder of this patent right is registered with ISO. Information can be obtained from:Agence Nationa
26、le de scurit sanitaire de lalimentation, de lenvironnement et du travail/ French Agency for Food, Environment and Occupational Health and Safety (ANSES)10 rue Pierre CurieF-94700 MAISONS-ALFORT, CedexFranceAttention is drawn to the possibility that some of the elements of this document may be the su
27、bject of patent rights other than those identified above. ISO shall not be held responsible for identifying any or all such patent rights.ISO (www.iso.org/patents) maintains an on-line database of patents relevant to their documents. Users are encouraged to consult the databases for the most up to d
28、ate information concerning patents. ISO 2012 All rights reserved vPD CEN ISO/TS 13136:2012Microbiology of food and animal feed Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens Horizontal method for the detection of Shiga toxin-producing Escherichia col
29、i (STEC) and the determination of O157, O111, O26, O103 and O145 serogroupsIMPORTANT It is necessary to consider any STEC as pathogenic to humans and potentially to cause severe disease depending on both the risk profile of the food commodity (ready-to-eat foods vs. foods intended to be consumed aft
30、er technological treatment such as pasteurization, cooking etc. used to reduce any bacteria present in the food) and the health status of the subject ingesting the food.Moreover, given the high genomic plasticity of this bacterial species, it is possible that novel arrangements of virulence features
31、 can give rise to novel sero-pathogroups such as the Shiga toxin-producing enteroaggregative E. coli O104 that caused the HUS outbreaks in Germany and France in 2011-05/06. Novel atypical E. coli sero-pathogroups can arise from the acquisition of an stx-converting bacteriophage by an E. coli strain
32、belonging to pathogroups different from STEC.Such atypical strains fall in the scope of this method and can be efficiently detected as they are positive for the presence of the stx genes.1 ScopeThis Technical Specification describes the identification of Shiga toxin-producing Escherichia coli (STEC)
33、 by means of the detection of the following genes:a) the major virulence genes of STEC, stx and eae (References 23);b) the genes associated with the serogroups O157, O111, O26, O103, and O145 (References 34).In any case, when one or both of the stx genes is/are detected, the isolation of the strain
34、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 tec
35、hnology for detection of the virulence and serogroup-associated genes.This Technical Specification 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
36、primary production.2 Normative referencesThe following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 7218,
37、 Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinationsTECHNICAL SPECIFICATION ISO/TS 13136:2012(E) ISO 2012 All rights reserved 1PD CEN ISO/TS 13136:2012ISO/TS 13136:2012(E)ISO 20838, Microbiology of food and animal feeding stuffs Polymeras
38、e chain reaction (PCR) for the detection of food-borne pathogens Requirements for amplification and detection for qualitative methodsISO 22174, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definition
39、s3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.Definitions 3.1 to 3.3 have been compiled from the epidemiological data on disease caused by STEC managed by organizations such as the US Centers for Disease Control and, in the EU, by the European C
40、entre for Disease Prevention and Control and the European Food Safety Authority.3.1Shiga toxin-producing Escherichia coliSTECE. coli strains possessing the Stx-coding genes3.2Shiga toxin-producing Escherichia coli causing the attaching and effacing lesionSTEC causing the attaching and effacing lesio
41、nE. coli strains possessing the Stx-coding genes and the intimin-coding gene eaeNOTE This combination of virulence genes is often associated with the most severe forms of STEC-induced disease.3.3Shiga toxin-producing Escherichia coli belonging to highly pathogenic serogroupsSTEC belonging to highly
42、pathogenic serogroupsE. coli strains possessing the Stx-coding genes, the intimin-coding gene eae and belonging to one of the serogroups O157, O111, O26, O103, and O1454 Principle4.1 GeneralThe method specified comprises the following sequential steps:a) microbial enrichment;b) nucleic acid extracti
43、on;c) detection of virulence genes;d) detection of serogroup-associated genes;e) isolation from positive samples.Figure A.1 is a flow diagram of the screening procedure.4.2 Microbial enrichmentThe number of STEC cells to be detected is increased by incubating the test portion in a non-selective liqu
44、id nutrient medium chosen from:a) modified tryptone-soy broth (tryptone soy broth supplemented with 1,5 g/l bile salts No.3, mTSB) supplemented with 16 mg/l of novobiocin (mTSB+N);b) buffered peptone water (BPW);2 ISO 2012 All rights reservedPD CEN ISO/TS 13136:2012ISO/TS 13136:2012(E)c) modified tr
45、yptone-soy broth (tryptone-soy broth supplemented with 1,5 g/l bile salts No.3, mTSB) supplemented with 12 mg/l of acriflavin (mTSB+A) for analysis of milk and dairy products.The mTSB shall be used when analysing matrices suspected to contain high levels of contaminating microflora. Novobiocin and a
46、criflavin inhibit the growth of Gram-positive bacteria and promote the growth of Gram-negative cells, including STEC. The BPW shall be used to analyse samples which are assumed to contain stressed target bacteria (such as frozen products), to resuscitate stressed STEC cells, and expected lower level
47、s of contaminating microflora than in fresh samples.NOTE The addition of novobiocin is controversial and has been investigated by several authors. It has been observed that the minimum inhibitory concentration of the antibiotic for non-O157 STEC is lower than for O157 strains (Reference 5). The addi
48、tion of novobiocin in the enrichment mTSB at the usual concentration of 20 mg/l, as specified in ISO 16654,19seems to inhibit the growth of about one-third of non-O157 strains (Reference 6) increasing the risk of false-negative results.4.3 Nucleic acid extractionThe nucleic acid is extracted accordi
49、ng to the requirements of the adopted detection system.4.4 Target genesThe purified nucleic acid is used for the detection of the following target genes: the main virulence genes for STEC: stx genes, encoding the Shiga toxins and the eae gene, encoding a 90 kDa protein, the intimin, involved in the attaching and effacing mechanism of adhesion, a typical feature of the STEC strains causing severe disease. The stx genes encode a family of toxins including two main types: stx1 and stx2. The latter compris
