1、 ISO 2017 Microbiology of food and animal feeding stuffs Horizontal method for the detection of Escherichia coli O157 AMENDMENT 1: Annex B: Result of interlaboratory studies Microbiologie des aliments Mthode horizontale pour la recherche des Escherichia coli O157 AMENDEMENT 1: Annexe B: Rsultat des
2、tudes interlaboratoires INTERNATIONAL STANDARD ISO 16654 First edition 2001-05-01 Reference number ISO 16654:2001/Amd.1:2017(E) AMENDMENT 1 2017-03 ISO 16654:2001/Amd.1:2017(E)ii ISO 2017 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2017, Published in Switzerland All rights reserved. Unless
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4、ess below or ISOs member body in the country of the requester. ISO copyright office Ch. de Blandonnet 8 CP 401 CH-1214 Vernier, Geneva, Switzerland Tel. +41 22 749 01 11 Fax +41 22 749 09 47 copyrightiso.org www.iso.org ISO 16654:2001/Amd.1:2017(E) Foreword ISO (the International Organization for St
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11、 .org/ iso/ foreword .html. This document was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/TC 275, Food analysis Horizontal methods, in collaboration with ISO Technical Committee TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the agr
12、eement on technical cooperation between ISO and CEN (Vienna Agreement). ISO 2017 All rights reserved iii Microbiology of food and animal feeding stuffs Horizontal method for the detection of Escherichia coli O157 AMENDMENT 1: Annex B: Result of interlaboratory studiesPage 8, 9.1 Replace “NOTE” with
13、“NOTE 1”.Page 8, 9.1 Add the following at the end of the subclause. This document has been validated for test portions of a specific weight or volume for each food matrix. A smaller test portion may be used, without the need for additional validation/verification, providing that the same ratio betwe
14、en (pre-) enrichment broth and test portion is maintained. A larger test portion than that initially validated may be used, if a validation/verification study has shown that there are no adverse effects on the detection of E. coli O157. NOTE 2 Validation can be conducted in accordance with the appro
15、priate documents of ISO 16140 (all parts). Verification for pooling samples can be conducted in accordance with the protocol described in ISO 6887-1:2017, Annex D (verification protocol for pooling samples for qualitative tests).Page 11, after Clause 11 Add the following as Clause 12 and renumber th
16、e previous Clause 12 “Test report” as Clause 13 “Test report”. 12 Performance characteristics of the method 12.1 Interlaboratory study The performance characteristics of the method were determined in interlaboratory studies to determine the specificity, sensitivity and, when possible, the LOD 50of t
17、he method. The data are summarized in Annex B. The values derived from the interlaboratory studies may not be applicable to food types other than those given in Annex B. 12.2 Sensitivity The sensitivity is the ability of the method to detect the analyte. It is defined as the number of samples found
18、positive divided by the number of samples tested at a given level of contamination. The results are thus dependent on the level of contamination of the sample. 12.3 Specificity The specificity is the ability of the method to correctly identify the absence of the analyte in negative samples. It is de
19、fined as the number of samples found negative divided by the number of blank samples tested. ISO 16654:2001/Amd.1:2017(E) ISO 2017 All rights reserved 1 ISO 16654:2001/Amd.1:2017(E) 12.4 LOD 50 The LOD 50(level of detection) is the concentration (cfu/sample) for which the probability of detection is
20、 50 %. The LOD 50has been calculated in the CEN Mandate studies (see Annex B) for the matrix sprouts only as the other study, on milk, returned no negative results for both the levels of contamination. The LOD 50was 8,4 cfu/g (6,5 cfu/g to 10,9 cfu/g confidence limit, 95 % confidence interval).Page
21、13, after Annex A Add the following as Annex B. Annex B (informative) Results of interlaboratory studies B.1 Results of interlaboratory studies of an NMKL study An international interlaboratory study was organized in 1999 by the Nordic Committee on Food Analysis on the following food matrices: mince
22、d meat, raw milk and lettuce (see Reference 1) on method assessed as equivalent to the method ISO 16654:2001. The values of the performance characteristics derived from this interlaboratory study are shown per type of sample in Tables B.1 to B.3. Table B.1 Results of data analysis obtained with 25 g
23、 minced meat samples Performance characteristic Minced meat (blank) Minced meat (low level, 17 cfu/sample) Minced meat (high level, 80 cfu/sample) Year of interlaboratory test 1999 1999 1999 Number of laboratories having returned results 14 14 14 Number of samples per laboratory 2 2 2 Number of excl
24、uded laboratories 0 0 0 Number of laboratories retained after the exclusion 14 14 14 Number of accepted samples 28 28 28 Sensitivity in % 100 100 Specificity in % 92,8 2 ISO 2017 All rights reserved ISO 16654:2001/Amd.1:2017(E) Table B.2 Results of data analysis obtained with 25 ml raw milk samples
25、Performance characteristic Raw milk (blank) Raw milk (low level, 17 cfu/sample) Raw milk (high level 78 cfu/sample) Year of interlaboratory test 1999 1999 1999 Number of laboratories having returned results 14 14 14 Number of samples per laboratory 2 2 2 Number of excluded laboratories 0 0 0 Number
26、of laboratories retained after the exclusion 14 14 14 Number of accepted samples 28 28 28 Sensitivity in % 92,8 100 Specificity in % 85,7 Table B.3 Results of data analysis obtained with 25 g lettuce samples Performance characteristic Lettuce (blank) Lettuce (low level, 10 cfu/sample) Lettuce (high
27、level, 33 cfu/sample) Year of interlaboratory test 1999 1999 1999 Number of laboratories having returned results 14 14 14 Number of samples per laboratory 1 2 2 Number of excluded laboratories 0 0 0 Number of laboratories retained after the exclusion 14 14 14 Number of accepted samples 14 28 28 Sens
28、itivity in % 100 100 Specificity in % 85,7 B.2 Results of interlaboratory studies of a CEN Mandate study B.2.1 Study on milk A second international interlaboratory study was run in 2012 in the framework of the CEN Mandate M/381. In the preliminary steps of the process, the method ISO 16654:2001 was
29、evaluated as being equivalent to the NMKL method N. 164 (See resolution 297 taken at the ISO/TC 34/SC 9 meeting in Prague in June 2006 for reference). Given the equivalence assessment between the two methods, at the 14th meeting of CEN/TC 275/WG 6 (23 to 28 April 2007, Cairo, Egypt) it was agreed to
30、 perform the validation study of the method ISO 16654:2001 in a reduced form, including only one epidemiologically relevant food matrix (milk). National reference laboratories (NRLs) for E. coli, and laboratories involved in the official controls of foodstuffs of EU Member States were selected based
31、 on whether they were accredited according to ISO 17025, had the method ISO 16654:2001 accredited and had expertise in IMS-based isolation of food-borne bacteria. In total, 15 laboratories participated in the inter-laboratory study, which was based on the analysis, with the method ISO 16654:2001, of
32、 raw milk samples artificially contaminated with E. coli O157 at three different levels of contamination: blank (0 cfu/ml), low (25 cfu/ml, combined uncertainty (Uc = 7 cfu/ml) and high (140 cfu/ml, Uc = 38 cfu/ml). The samples contained naturally present background microflora, and eight blind repli
33、cates of each level of contamination were sent to each laboratory, for a total of 24 samples examined. The samples contained glycerol and were sent to the laboratories as frozen specimens (70 C in dry ice). ISO 2017 All rights reserved 3 ISO 16654:2001/Amd.1:2017(E) The values of the performance cha
34、racteristics derived from this collaborative test is shown in Table B.4. Table B.4 Results of data analysis obtained with 10 ml milk samples Performance characteristic Milk (blank) Milk (low level, 250 cfu/sample) Milk (high level, 1 400 cfu/sample) Year of interlaboratory test 2012 2012 2012 Number
35、 of laboratories having returned results 15 15 15 Number of samples per laboratory 8 8 8 Number of excluded laboratories 0 0 0 Number of laboratories retained after the exclusion 15 15 15 Number of accepted samples 120 120 120 Sensitivity in % 100 100 Specificity in % 94,4 B.2.2 Study on sprouts A t
36、hird international interlaboratory study was organized and run in 2013 involving laboratories enrolled according to the same principles used for the first CEN mandate study on milk. Fourteen laboratories received a total of 24 samples consisting of 8 blind replicates of sprouts, purchased at retail,
37、 spiked with 3 different concentrations of E. coli O157: 0 cfu/g, 10 cfu/g and 100 cfu/g. The extended uncertainty of measurement associated with the inoculum was 0,27 log cfu/ml (calculated according to the ISO/TS 19036). The sprout samples each contained a mixture of alfalfa (90 %) and watercress
38、(10 %), with an endogenous microflora estimated to be 10 5cfu/g to 10 7cfu/g and have been sent refrigerated to the participating laboratories. The values of the performance characteristics derived from this collaborative test are shown in Table B.5. Table B.5 Results of data analysis obtained with
39、25 g sprout samples Performance characteristic Sprouts (blank) Sprouts (low level, 250 cfu/sample) Sprouts (high level, 2 500 cfu/sample) Year of interlaboratory test 2014 2014 2014 Number of laboratories having returned results 14 14 14 Number of samples per laboratory 8 8 8 Number of excluded labo
40、ratories 0 0 0 Number of laboratories retained after the exclusion 14 14 14 Number of accepted samples 112 112 112 Sensitivity in % 75,9 96,4 Specificity in % 99,1 Bibliography Add the following reference. 7 NMKL N. 164:1999, Escherichia coli O157. Detection in food and feeding stuffs4 ISO 2017 All rights reserved ISO 16654:2001/Amd.1:2017(E) ISO 2017 All rights reserved IC S 07.100.30 Price based on 4 pages
