EN ISO 24276-2006 en Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - General requirements and definitions (Incorporates .pdf

1、%5,7,6+67$1$5%6(1,62)RRGVWXIIV0HWKRGVRIDQDOVLVIRUWKHGHWHFWLRQRIJHQHWLFDOOPRGLILHGRUJDQLVPVDQGGHULYHGSURGXFWV*HQHUDOUHTXLUHPHQWVDQGGHILQLWLRQV7KH(XURSHDQ6WDQGDUG(1,62KDVWKHVWDWXVRID%ULWLVK6WDQGDUG, grinding of the test sample; preparation of test portions; extraction of the analyte; testing, interpre

2、tation, and reporting of the results.Procedure-specific instructions are found within the main text and the individual annexes of ISO 21569, ISO 21570, ISO 21571 and ISO 21572.5.2 Use of controlsThe controls shall be used according to Table 1. Table 1 is applicable to DNA-based methods only. Control

3、s applicable to protein-based methods are described in ISO 21572.ISO 24276:2006/Amd.1:2013(E)Table 1 Flow diagram showing intersection of successive steps and inclusion of controlsControl stepEnviron- ment con-trolbExtraction blank con-trolcPositive extraction controldPositive DNA target controleNeg

4、ative DNA target controlfAmplifica-tion reagent controlgPCR inhibi-tion controlhHomogeniza-tionMandatory Nucleic acid extractionaOne per seriesMandatory at regular intervals Assessment of nucleic acid quality Nucleic acid amplification MandatoryRecom-mendedMandatoryRecommen- ded, but mandatory in ce

5、rtain casesiAssessment of results of nucleic acid amplification Interpreta-tion Test repor t aThe arrows indicate that this control should be applied in the subsequent analytical steps.bThe use of environment controls helps the laboratory to identify sources of contamination at an early stage and ca

6、n even be used to identify in which work area the contamination is present. This can be demonstrated in various ways, e.g. if negative samples included in the series of homogenized samples showed negative results, starting at the first step of the process (e.g. grinding step if relevant)cAt least on

7、e extraction blank control shall be included each time DNA is extracted from one or more samples. The tube shall always be the last in each series. It may be appropriate to put one extraction blank on, for example, a rack of eight tubes or a microplate of 96 wells for automated extraction.dA positiv

8、e extraction control should be included regularly, and always when a new batch of extraction reagents is used. This control reveals if something is wrong with the reagents or the performance of the extraction protocol.eThe positive DNA target control demonstrates the ability of the nucleic acid ampl

9、ification procedure to detect the nucleic acid representative of the GMO or target taxon. This condition can also be fulfilled by an appropriate positive extraction control.fThe negative DNA target control demonstrates the ability of the nucleic acid amplification procedure to avoid false positive a

10、mplification in the absence of the nucleic acid representative of the GMO or target taxon.gThe amplification reagent control demonstrates the absence of contaminating nucleic acid in the PCR reagent batches used. The amplification reagent control can be omitted when the extraction blank control is u

11、sed.hThe PCR inhibition control may be used to demonstrate the absence of soluble inhibitors. This may also be demonstrated by serial dilutions of the template nucleic acid. However, some type of assessment of the effect of soluble inhibitors on the results of the analysis of the sample shall be mad

12、e.iA PCR-inhibition control is mandatory, if all PCR-tests on the sample are negative and for matrices where the yield of amplifiable DNA is not known.Page 12, 5.3.4Replace the existing text with the following.The laboratory should use properly maintained equipment suitable for the methods employed.

13、 In addition to standard laboratory equipment, additional apparatus is described in the annexes of the specific standards.Apparatus and equipment shall be maintained according to guidelines outlined in ISO/IEC 17025;10manufacturers instructions should be taken into consideration.Page 14, Table 2Repl

14、ace the existing Table 2 with the following.8 ISO 2013 All rights reservedBS EN ISO 24276:2006+A1:2013ISO 24276:2006+A1:201311 /DERUDWRU RUJDQLDWLRQ *HQHUDO10 manufacturers instructions should be taken into consideration. 0DWHULDOV DQG UHDJHQWV)RUWKH DQDOVLVXQOHVV RWKHUZLVH VWDWHGXVH RQO DQDOWLFDO J

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19、ISO 24276:2006+A1:201314 ISO 24276:2006/Amd.1:2013(E)Table 2 Examples of PCR resultsTest sam-plePositive extrac-tion controlExtraction blank controlNegative DNA tar-get controlPositive DNA target controlInterpreted result+a+ b+ positive + + negative+ + + + inconclusivec + inconclusivec inconclusived

20、aPCR product is detectable within the detection limit of analytical method used and test portion analysed.bNo PCR product is detectable within the detection limit of analytical method used and test portion analysed.cThe procedure shall be repeated beginning with the extraction step (possible contami

21、nation).dThe procedure shall be repeated using another extraction method or a further purification step (possible inhibition).Page 14, 6.3 to 6.5Replace the existing text with the following.6.3 Expression of a negative resultThe following text shall appear in the test report:“For sample X, target se

22、quence Y was not detected.The LOD of the method is x % determined with ABC (identify the reference material).”If it cannot be demonstrated that the amount of target DNA included in the PCR is sufficient for the LOD to be applicable, then the following sentence shall be added:“However, the amount of

23、the target DNA extracted from species X may be/was insufficient for the LOD to be applicable for this sample.”In addition, if applicable: “The practical limit of detection is x %”.NOTE The practical LOD of the sample is determined by the quantity of DNA of the species included in the analytical reac

24、tion (copy number), and the ratio relative to the absolute LOD of the GM target (copy number).6.4 Expression of a positive resultIn case of a qualitative analysis, the following text shall appear in the test report:“For sample X, target sequence Y was detected.”The identity of the GMO may be include

25、d, if available.In case of a quantitative analysis: if the target taxon-specific sequence and the GM target sequence are both detected but the quantity is below the LOQ of at least one of the target sequences, the following text shall appear in the test report for each GMO sequence:“GMO (specify the

26、 GMO) derived DNA as determined by detection of (specify target sequence) derived from (specify species) was detected, below the practical limit of quantification” ISO 2013 All rights reserved 9ISO 24276:2006/Amd.1:2013(E)Table 2 Examples of PCR resultsTest sam-plePositive extrac-tion controlExtract

27、ion blank controlNegative DNA tar-get controlPositive DNA target controlInterpreted result+a+ b+ positive + + negative+ + + + inconclusivec + inconclusivec inconclusivedaPCR product is detectable within the detection limit of analytical method used and test portion analysed.bNo PCR product is detect

28、able within the detection limit of analytical method used and test portion analysed.cThe procedure shall be repeated beginning with the extraction step (possible contamination).dThe procedure shall be repeated using another extraction method or a further purification step (possible inhibition).Page

29、14, 6.3 to 6.5Replace the existing text with the following.6.3 Expression of a negative resultThe following text shall appear in the test report:“For sample X, target sequence Y was not detected.The LOD of the method is x % determined with ABC (identify the reference material).”If it cannot be demon

30、strated that the amount of target DNA included in the PCR is sufficient for the LOD to be applicable, then the following sentence shall be added:“However, the amount of the target DNA extracted from species X may be/was insufficient for the LOD to be applicable for this sample.”In addition, if appli

31、cable: “The practical limit of detection is x %”.NOTE The practical LOD of the sample is determined by the quantity of DNA of the species included in the analytical reaction (copy number), and the ratio relative to the absolute LOD of the GM target (copy number).6.4 Expression of a positive resultIn

32、 case of a qualitative analysis, the following text shall appear in the test report:“For sample X, target sequence Y was detected.”The identity of the GMO may be included, if available.In case of a quantitative analysis: if the target taxon-specific sequence and the GM target sequence are both detec

33、ted but the quantity is below the LOQ of at least one of the target sequences, the following text shall appear in the test report for each GMO sequence:“GMO (specify the GMO) derived DNA as determined by detection of (specify target sequence) derived from (specify species) was detected, below the pr

34、actical limit of quantification” ISO 2013 All rights reserved 9 ISO 24276:2006/Amd.1:2013(E)Table 2 Examples of PCR resultsTest sam-plePositive extrac-tion controlExtraction blank controlNegative DNA tar-get controlPos tive DNA target controlInterpreted result+a+ b+ positive + + negative+ + + + inco

35、nclusivec + inconclusivec inconclusivedaPCR product is d tectable within the d tection limit of analytical method used and test portion analysed.bNo PCR product is d tectable within the d tection limit of analytical method used and test portion analysed.cThe procedure shall be repeated begi ing with

36、 the extraction step (possible contamination).dThe procedure shall be repeated using another extraction method or a further pur fication step (possible inhib tion).Page 14, 6.3 to 6.5Replace the existing text with the following.6.3 Expre sion of a negative resultThe following text shall a pear in th

37、e test report:“For sample X, target sequence Y was not detected.The LOD of the method is x % determined with ABC (identify the reference material).”If it ca not be demonstrated that the amount of target DNA included in the PCR is sufficient for the LOD to be a plicable, then the following sentence s

38、hall be a ded:“However, the amount of the target DNA extracted from species X may be/was insufficient for the LOD to be a plicable for this sample.”In a dition, if a plicable: “The practical limit of detection is x %”.NOTE The practical LOD of the sample is determined by the quantity of DNA of the s

39、pecies included in the analytical reaction (copy number), and the ratio relative to the absolute LOD of the GM target (copy number).6.4 Expre sion of a positive resultIn case of a qualitative analysis, the following text shall a pear in the test report:“For sample X, target sequence Y was detected.”

40、The identity of the GMO may be included, if available.In case of a quantitative analysis: if the target taxon-spec fic sequence and the GM target sequence are both detected but the quantity is below the LOQ of at least one of the target sequences, the following text shall a pear in the test report f

41、or each GMO sequence:“GMO (specify the GMO) derived DNA as determined by detection of (specify target sequence) derived from (specify species) was detected, below the practical limit of quant fication” ISO 2013 All rights reserved 9 4XDOLW DVVXUDQFH UHTXLUHPHQWV*HQHUDO DVSHFWV RI TXDOLW DVVXUDQFH DU

42、H FRYHUHG LQ ,62,(b) any particular information relating to the laboratory sample;c) All information related to the test sample (size of the sample ground);d) reference to this International Standard (ISO 24276:2006) and the relevant annex(es) followed;e) statement about date and type of sampling pr

43、ocedure(s) used;f) date of receipt;g) storage conditions, if applicable;h) analysis start and end dates, if applicable;i) person responsible for the analysis;j) results according to the requirements of the specific method and the units used to report the results and the calibrators and the calculation method used;k) any particular observations made during testing;l) any deviations, additions to, or exclusions from, the test specification;m) r