1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationPD CEN ISO/TS 15216-2:2013Microbiology of food andanimal feed Horizontalmethod for determination ofhepatitis A virus and norovirusin food using real-time RT-PCRPart 2: Method for
2、 qualitative detectionPD CEN ISO/TS 15216-2:2013 PUBLISHED DOCUMENTNational forewordThis Published Document is the UK implementation ofCEN ISO/TS 15216-2:2013.The UK participation in its preparation was entrusted to TechnicalCommittee AW/9, Microbiology.A list of organizations represented on this co
3、mmittee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2013. Published by BSI StandardsLimited 2013 ISBN 978 0 580 80627 8 ICS 07.10
4、0.30 Compliance with a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of theStandards Policy and Strategy Committee on 31 May 2013. Amendments issued since publicationDate T e x t a f f e c t e dTECHNICAL SPECIFICATION SPCIFICA
5、TION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN ISO/TS 15216-2 May 2013 ICS 07.100.30 English Version Microbiology of food and animal feed - Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR - Part 2: Method for qualitative detection (ISO/TS 15216-2:20
6、13, Corrected Version 2013-05-01) Microbiologie des aliments - Mthode horizontale pour la recherche des virus de lhpatite A et norovirus dans les aliments par la technique RT-PCR en temps rel - Partie 2: Mthode de dtection qualitative (ISO/TS 15216-2:2013, Version Corrige 2013-05-01) Mikrobiologie v
7、on Lebensmitteln und Futtermitteln - Horizontales Verfahren zum Nachweis von Hepatitis A-Viren und Noroviren in Lebensmitteln mittels Real time PCR - Teil 2: Verfahren fr den qualitativen Nachweis (ISO/TS 15216-2:2013, korrigierten Fassung von 2013-05-01) This Technical Specification (CEN/TS) was ap
8、proved by CEN on 8 March 2013 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 a European Standa
9、rd. 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 the final decisio
10、n 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, Iceland, Ireland, Ita
11、ly, 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: Avenue Marnix 1
12、7, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN ISO/TS 15216-2:2013: EPD CEN ISO/TS 15216-2:2013CEN ISO/TS 15216-2:2013 (E) 3 Foreword This document (CEN ISO/TS 15216-2:2013) has been prepared by Technical
13、Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is held by DIN. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENE
14、LEC 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 announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech R
15、epublic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. E
16、ndorsement notice The text of ISO/TS 15216-2:2013, Corrected Version 2013-05-01 has been approved by CEN as CEN ISO/TS 15216-2:2013 without any modification. PD CEN ISO/TS 15216-2:2013ISO/TS 15216-2:2013(E) ISO 2013 All rights reserved iiiContents PageForeword ivIntroduction vii1 Scope . 12 Normativ
17、e references 13 Terms and definitions . 14 Principle 34.1 Virus extraction 34.2 RNA extraction 34.3 Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) . 34.4 Control materials 44.5 Test results 45 Reagents 45.1 General . 45.2 Reagents used as supplied 45.3 Prepared reagents
18、 66 Apparatus and materials 67 Sampling 88 Procedure. 88.1 General laboratory requirements . 88.2 Virus extraction 88.3 RNA extraction . 108.4 Real-time RT-PCR . 109 Interpretation of results 129.1 General 129.2 Construction of process control virus RNA standard curve129.3 Control for amplification
19、efficiency . 129.4 Calculation of extraction efficiency 139.5 Theoretical limit of detection 1310 Expression of results .1311 Test report 14Annex A (normative) Diagram of procedure .15Annex B (informative) Real-time RT-PCR mastermixes and cycling parameters 16Annex C (informative) Real-time RT-PCR p
20、rimers and hydrolysis probes for the detection of HAV, norovirus GI and GII and mengo virus (process control) 17Annex D (informative) Growth of mengo virus strain MC0for use as a process control .19Annex E (informative) RNA extraction using the BioMerieux NucliSenssystem .20Annex F (normative) Compo
21、sition and preparation of reagents and buffers 22Annex G (informative) Generation of external control RNA (EC RNA) stocks .24Annex H (informative) Typical optical plate layout .26Bibliography .27PD CEN ISO/TS 15216-2:2013ISO/TS 15216-2:2013(E)ForewordISO (the International Organization for Standardi
22、zation) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to b
23、e represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.International Standar
24、ds are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an Interna
25、tional Standard requires approval by at least 75 % of the member bodies casting a vote.In other circumstances, particularly when there is an urgent market requirement for such documents, a technical committee may decide to publish other types of document: an ISO Publicly Available Specification (ISO
26、/PAS) represents an agreement between technical experts in an ISO working group and is accepted for publication if it is approved by more than 50 % of the members of the parent committee casting a vote; an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technica
27、l committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a vote.An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further three years, revised to become an International Standard, or withdrawn.
28、 If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an International Standard or be withdrawn.Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. I
29、SO shall not be held responsible for identifying any or all such patent rights.ISO/TS 15216-2 was prepared by the European Committee for Standardization (CEN), in collaboration with Technical committee ISO/TC 34, Food products, Subcommittee SC 9 Microbiology.This corrected version of ISO/TS 15216-2:
30、2013 incorporates the following corrections. Throughout, textual references have been updated to take reordering of the annexes into account. Annex B was formerly Annex E; Annex C was formerly Annex D; Annex D was formerly Annex G; Annex E was formerly Annex C; Annex F was formerly Annex B; Annex G
31、was formerly Annex H; Annex H was formerly Annex F. Where units of shaking operations are mentioned, “oscillations min1” replaces “min1”. Many cross-references to reagents or apparatus subclauses are added. A phrase citing Annex A is added to the end of the introduction. The definitions for “food su
32、rface” (formerly 3.2 and 3.3) are combined and expanded in a redrafted 3.2; in consequence, the following terms in Clause 3 are renumbered. In 3.4, Note 2, “There is only one serotype” is transposed to the end of Note 1. Also, “group 2 biological agent by the European Union and as a risk group 2 hum
33、an aetiological agent by the United States National Institutes of Health” replaces “UK Advisory Committee on Dangerous Pathogens (ACDP) hazard group 2 pathogen”.iv ISO 2013 All rights reservedPD CEN ISO/TS 15216-2:2013ISO/TS 15216-2:2013(E) In 3.5, Note 2, “group 2 biological agents by the European
34、Union and as risk group 2 human aetiological agents by the United States National Institutes of Health” replaces “ACDP hazard group 2 pathogens”. In 3.13, “used in” replaces “used as template in”. In 5.2.11, “from Aspergillus niger or A. aculeatus” is inserted after “Pectinase”. In 6.1,”Aerosol resi
35、stant tips should be used unless unobstructed tips are required, e.g. for aspiration.” is inserted. In 6.5, “37 1,0” replaces “37 10”. A redrafted 6.10 on centrifuge(s) and rotor(s) replaces the former 6.10 and 6.11, with consequent renumbering of the following subclauses. In 6.19, the square bracke
36、ts are deleted. In 6.27, “Real-time PCR machine(s), i.e. thermal cycler(s),” replaces “Thermal cycler(s)”. In 6.28, “selected real-time PCR” replaces “selected PCR”. In 8.1, “Samples arriving already frozen should be defrosted prior to testing.” is inserted as the second sentence. 8.2.3 Is redrafted
37、. In 8.2.4, paragraph 2,”buffer (5.3.5) (for soft fruit samples, add 30 units pectinase from A. niger, or 1 140 units pectinase from A. aculeatus to the buffer) and” replaces “buffer (for soft fruit samples, add 30 units pectinase to the buffer) and”. In 8.2.6, paragraph 2, “and the animal is suppor
38、ted with a rubber block” is added. In 8.2.6, last paragraph, “min at room temperature, decant” replaces “min, decant” In 8.4.2.3, paragraph 1,”using a real-time PCR machine (6.27)” is added. In 9.4, Note 1 “has a process control virus recovery (equal to the extraction efficiency in matrices other th
39、an BMS) of 100 %. For a process control virus RNA standard curve with an idealized slope of 3,32, if the Cqvalue of an undiluted sample RNA well is 1% and therefore acceptable” replaces “will have an extraction efficiency of 100 %”. The title of Annex B has been expanded to read, “Real-time RT-PCR m
40、astermixes and cycling parameters”. In Table B.1, footnote a, “real-time PCR machines” twice replaces “real-time machines”. In C.1, “This primer set amplifies a product of 173 bp corresponding to nucleotides 68240 of HAV isolate HM174 43c (GenBank accession number M59809).” is added as paragraph 2.
41、In C.2, “This primer set amplifies a product of 86 bp corresponding to nucleotides 52915376 of Norwalk virus (GenBank accession number M87661).” is added as paragraph 2. In C.3, “This primer set amplifies a product of 89 bp corresponding to nucleotides 50125100 of Lordsdale virus (GenBank accession
42、number X86557).” is added as paragraph 2. In C.4, “This primer set amplifies a product of 100 bp corresponding to nucleotides 110209 of the deletant mengo virus strain MC0used in the development of this part of ISO/TS 15216. This corresponds to nucleotides 110270 of the non-deletant mengo virus isol
43、ate M (GenBank accession number L22089).” is added as paragraph 2. ISO 2013 All rights reserved vPD CEN ISO/TS 15216-2:2013ISO/TS 15216-2:2013(E) In G.5, “mastermix (if the Cqdifference between EC RNA stock tested with heat-treated and untreated mastermix is 1% and therefore acceptable.NOTE 2 A samp
44、le showing an unacceptable extraction efficiency, but producing an otherwise valid positive result can, if appropriate, be reported as positive as described in Clause 10.9.5 Theoretical limit of detectionThe theoretical limit of detection (tLOD) is the level that constitutes the smallest quantity of
45、 target that is in theory be detected. This corresponds to one genome copy per volume of RNA tested in the target assay, but varies according to the test matrix and the quantity of starting material.For each hard surface, soft fruit, salad vegetable or bottled water sample, the minimum quantity of t
46、arget in the entire sample that is detectable in theory is 20 (results using undiluted sample RNA) or 200 genome copies (101RNA).For BMS samples, the theoretical minimum quantity detectable is obtained by dividing the above values by 0,5 and multiplying by the total homogenate volume.To obtain the t
47、LOD of each sample in detectable virus genome copies per millilitre, per gram or per square centimetre, divide the number of theoretically detectable genome copies in the entire sample by the starting volume (bottled water), mass (BMS, soft fruits, salad vegetables) or area (hard surfaces) of the sa
48、mple.10 Expression of resultsPositive results for each target virus for food surfaces shall be expressed as “virus genome detected”; for other sample types, positive results shall be expressed as “virus genome detected in x ml” or “virus genome detected in x g”where x is the amount of sample tested.
49、 ISO 2013 All rights reserved 13PD CEN ISO/TS 15216-2:2013ISO/TS 15216-2:2013(E)If target virus is not detected, results for food surfaces shall be expressed as “virus genome not detected”; for other sample types, results shall be expressed as “virus genome not detected in x ml” or “virus genome not detected in x g”.If a valid result is not obtained, results shall normally be expressed as “no result”. If however, an otherwise valid positive result is obtained from a sample showing an unacceptable amplification or e
