1、ICS 07.100.30Nachweis von Salmonellen in Lebensmitteln mittels enzymgebundenen FluoreszenzimmunoassayIn keeping with current practice in standards published by the International Organization for Standardization(ISO), a comma has been used throughout as the decimal marker.ForewordThis standard has be
2、en prepared by Technical Committee Mikrobiologische Lebensmitteluntersuchungeinschlielich Schnellverfahren of the Normenausschuss Lebensmittel und landwirtschaftliche Produkte(Foodstuffs and Agricultural Products Standards Committee).Ref. No. DIN 10121 : 2000-08English price group 07 Sales No. 01070
3、1.02DEUTSCHE NORM August 200010121Continued on pages 2 to 4. No part of this translation may be reproduced without the prior permission ofDIN Deutsches Institut fr Normung e.V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).Detecti
4、on of salmonella in foodstuffs byenzyme-linked fluorescent immunoassayTranslation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.1 ScopeThis standard specifies a routine method of detecting salmonella by an enzyme-linked fluorescent
5、 immu-noassay (ELFA).NOTE: Attention is drawn to the stipulations of the Gesetz zur Verhtung und Bekmpfung bertragbarerKrankheiten beim Menschen) (German Act on the prevention and control of contagious human diseases).2 Normative referencesThis standard incorporates, by dated or undated reference, p
6、rovisions from other publications. Thesenormative references are cited at the appropriate places in the text, and the titles of the publications arelisted below. For dated references, subsequent amendments to or revisions of any of these publicationsapply to this standard only when incorporated in i
7、t by amendment or revision. For undated references, thelatest edition of the publication referred to applies.DIN EN 12824 Microbiology of food and animal feeding stuffs Horizontal method for the detection ofsalmonella (modified version of ISO 6579 : 1993)ISO 7218 : 1996 Microbiology of food and anim
8、al feeding stuffs General rules for microbiological exami-nationGesetz zur Verhtung und Bekmpfung bertragbarer Krankheiten beim Menschen (Bundesseuchengesetz),BGBl. (German Federal Law Gazette) I, 1961, No. 53, pp. 1012-10293 Concepts3.1 SalmonellaMicroorganisms that are bound by monoclonal salmonel
9、la antibodies and detected by a fluorescent reac-tion. They form typical colonies on solid selective media and display the biochemical and serologicalcharacteristics described when testing in accordance with DIN EN 12824.3.2 Detection of salmonellaDetection of the presence or absence of salmonella,
10、in a particular mass of product, by the method specifiedin this standard.Page 2DIN 10121 : 2000-084 Principle4.1 GeneralThe detection of salmonella involves several successive stages.If commercially available immunological ELFA/ELISA test systems are used that yield results comparable tothose obtain
11、ed when using this standard, the detection shall always be performed as specified by the manu-facturer. The method described here is given by way of example.NOTE: Since salmonella may occur in sample material in small numbers, in sublethally damaged form ortogether with a larger number of other micr
12、oorganisms, a pre-enrichment and enrichment in a selective liquidmedium are necessary.4.2 Pre-enrichment in a non-selective liquid mediumBuffered peptone water is inoculated with the sample and incubated at 37 C for 16 hours to 20 hours.4.3 Enrichment in selective liquid mediaA Rappaport-Vassiliadis
13、 magnesium chloride/malachite green medium and a selenite/cystine medium areinoculated with the culture obtained as described in subclause 4.2 and incubated at 42 C and 37 C for 6 hoursto 8 hours, respectively.4.4 Incubation after enrichment in a non-selective liquid mediumMannose broth is inoculate
14、d with the cultures obtained as described in subclause 4.3 and incubated at 42 Cfor 18 hours; then, 1 ml of each broth is heated at 100 C for 15 minutes in a water bath.4.5 Detection of salmonellaThe suspensions obtained as described in subclause 4.4 are brought together with salmonella antibodies i
15、n anantibody carrier and, after binding by an antigen-antibody reaction, the salmonella are detected by a fluorescentreaction.4.6 Confirmation and identificationIf the result of detection performed as in subclause 4.5 is positive, the portion of broth not heated as insubclause 4.4 and the magnesium
16、chloride/malachite green and selenite/cystine media are used for confirma-tion. Two selective solid media are inoculated with these cultures. Then the procedures described in subclauses4.3 and 4.4 of EN 12824, February 1998 edition, are followed.5 Culture media, reagents and sera5.1 GeneralIn additi
17、on to standard laboratory aids as described in ISO 7218, the following shall be used.5.2 Culture media and reagents5.2.1 GeneralAll the culture media and reagents specified in appendices B.1 to B.13 of DIN EN 12824 will be required.5.2.2 Mannose broth5.2.2.1 Composition Yeast extract 5,0 g Pancreati
18、cally digested casein peptone 12,5 g D-mannose 2,0 g Sodium citrate 5,0 g Sodium chloride 5,0 g Dipotassium phosphate 5,0 g Magnesium sulfate 0,8 g Manganese chloride 0,14 g Iron(III) sulfate 0,04 g Polyoxyethylene sorbitan monolaurate (e.g. Tween 80)1)75g Water 1000 ml1) Tween 80is the trade name o
19、f a product supplied by ICI-America Inc. This information is given for theconvenience of users of this standard and does not constitute an endorsement of the product by DIN.Equivalent products may be used if they can be demonstrated to give identical results.Page 3DIN 10121 : 2000-085.2.2.2 Preparat
20、ionDissolve the constituents in water and boil for one to two minutes at 100 C. Adjust the pH value to 7,0 t 0,2at 25 C, transfer 10 ml aliquots to culture tubes and autoclave at (121 t 1) C for 15 minutes.5.3 SeraSee subclause 5.3 of DIN EN 12824.5.4 Reagents and materials for immunoassay5.4.1 Anti
21、body carrier2), coated with monoclonal antibodies specific to salmonella surface antigen.5.4.2 Cuvettes5.4.2.1 Sampling cuvette, for 500 l of enrichment medium as in subclause 4.4.5.4.2.2 Pre-wash buffer and wash buffer prepared by combininga) 400 l of 18,15 g/l (150 mmol/l) tris NaCl polyoxyethylen
22、e sorbitan monolaurate (e.g. Tween 80)1) andb) 600 l of 1 g/l sodium azide.5.4.2.3 Conjugate400 l of conjugate, prepared by adding 1 g/l sodium azide to polyclonal salmonella antibodies (mutton, goat)labelled with alkaline phosphatase.5.4.2.4 Substrate, prepared by introducing 300 l of 4-methylumbel
23、liferyl phosphate and 1 g/l sodium azideinto a 500 l cuvette.5.4.2.5 Positive controlPurified and inactivated salmonella antigen with 1 g/l sodium azide.5.4.2.6 Negative controlTris sodium chloride polyoxyethylene sorbitan monolaurate (e.g. Tween 80)1) with 1 g/l sodium azide.5.4.2.7 Standard antige
24、n solutionPurified and inactivated salmonella antigen with 1 g/l sodium azide of known concentration.6 ApparatusIn addition to standard microbiological laboratory equipment and the apparatus specified in clause 6 ofDIN EN 12824, the following shall be used.6.1 Water bath, capable of operating at (10
25、0 t 1) C.6.2 Photocell, for fluorescence measurement at wavelengths around 450 nm.6.3 Pipettes, of capacity 100 l to 1000 l and pipette tips.7 SamplingAs in clause 7 of DIN EN 12824.8 Sample preparationAs in clause 8 of DIN EN 12824.9 Procedure9.1 Initial sample mass and first dilutionAs in subclaus
26、e 9.1 of DIN EN 12824.9.2 Non-selective pre-enrichmentAs in subclause 9.2 of DIN EN 12824.1) See page 2.2) Information on sources of supply is obtainable from the Normenausschuss Lebensmittel und landwirtschaft-liche Produkte of DIN, Burggrafenstr. 6, 10787 Berlin, Germany.Page 4DIN 10121 : 2000-089
27、.3 Selective enrichmentAs in subclause 9.3 of DIN EN 12824, with the following modification.Inoculate 10 ml of a magnesium chloride/malachite green medium with 0,1 ml of the culture obtained asspecified in subclause 9.2 and incubate at 42 C for 6 hours to 8 hours. Inoculate 10 ml of a selenite/cysti
28、nemedium with 1 ml of the culture obtained as specified in subclause 9.2 and incubate at 37 C for six to eighthours.9.4 Incubation after enrichment in a non-selective liquid mediumIntroduce 1 ml of each of the cultures obtained as specified in subclause 9.3 in magnesium chloride/malachitegreen mediu
29、m and in selenite/cystine medium into one tube in each case containing mannose broth andincubate at 42 C and 37 C, respectively, for 18 hours. After incubation, introduce 1 ml of each of the culturesobtained in the two broths together into a tube and mix. Heat the suspension for 15 minutes in the wa
30、ter bathat 100 C. Store the remainder of the culture medium and of the selective media at 4 C to confirm a positiveresult.If the incubated broth cannot be processed immediately, it may be stored at 4 C for 48 hours, but longerstorage will require freezing. Heating at 100C shall be carried out after
31、storage.9.5 Detection of salmonellaAll the reagents and materials shall be conditioned at ambient temperature for at least 30 minutes before use.Reagents belonging to different batches shall not be mixed. All the reagents and solutions shall be thoroughlymixed before use.Introduce 500 l of the heate
32、d culture obtained as specified in subclause 9.4 in a cuvette and then immerse theantibody carrier coated with salmonella antigen antibody into the cuvette. If calibration as in clause 12 is to becarried out, immerse two antibody carriers coated with positive and negative antigen controls at the sam
33、e timein the cuvette and measure twice using the antigen standard solution specified in subclause 5.4.2.7.Following this, immerse the respective antibody carriers once in pre-wash buffer and six times in wash buffer.Then, immerse the respective antibody carriers in the cuvette containing conjugate a
34、nd then, in the cuvettecontaining substrate.Measure the colour reaction produced fluorometrically, always performing a duplicate measurement. The firstmeasurement serves to determine the background value by measuring a cuvette containing substrate withoutfurther additives, the second measurement bei
35、ng performed after immersing the antibody carriers in the cuvettecontaining substrate. The difference between the two values yields a relative fluorescence value (RFV). The testvalue used for assessment is given by dividing the RFV of the sample by that of the antigen standard solution.9.6 Confirmat
36、ion and isolationRegard a relative fluorescence value less than 0,23 as negative, i.e. as a sample not containing detectablesalmonella antigens. Regard a value of greater than or equal to 0,23 as positive, i.e. salmonella antigens havebeen detected. To isolate the salmonella from the sample, use the
37、 previously stored broth and the incubatedselective media.Carry out the subsequent subculturing on solid culture media and the confirmation reactions as described insubclauses 9.4 and 9.5 of DIN EN 12824.10 EvaluationAs specified in clause 10 of DIN EN 12824.11 Test reportAs specified in clause 11 o
38、f DIN EN 12824.If an automated method is used, specify the system and the manufacturers reagent batch designations. Afterautomated evaluation in the system, batch number and fluorescence values of the standard and samples willbe printed out.12 Quality controlIn addition to the procedure specified in
39、 clause 12 of DIN EN 12824, every time new reagent batches are useda positive and negative control shall be included, as described in this standard. Where a new batch has not beenused for two weeks, a calibration shall be performed with the standard solution using pipetted (500 t 5) lsample. If the
40、values found for the positive control and the standard are not within the previously determinedrange, the sample results shall not be used and the assay is to be repeated using a fresh control solution.If the background value of the substrate sample is above a specified value, indicating contamination of thesolution, the assay shall be repeated with a fresh solution.
