AOAC ERP MICRO AUGUST 2018

Table 989.14B. Interlaboratory study results for determination of Salmonella in all foods by 3M TECRA Salmonella Visual Immunoassay (based on interlaboratory study results of the original method)

Test portions positive

c 2 b

c 2

Test portion analyzed a

BAM/AOAC

EIA

EIA confirmed

Product Pepper

96

64 90

63 90

0.00

63 90

0.00

— c

Soy isolate

108 120

— — —

Dried whole egg

100

100

—

100

Chocolate

96

79 79 73

80 78 82

0.00 0.00 0.75

79 78 77

Nonfat dry milk

108 118

0.00 0.31

Turkey

a Does not include test portions eliminated because of outlier data or method deviations. b McNemar test c 2 value >3.84 indicates significance ( P <0.05). c Statistical analysis not applicable. Methods gave identical results.

( f ) Enzyme immunoassay reader or dual wavelength reader with filters at 414 ± 10 nm and 490 ± 10 nm .—Optional. Photometer with 414 ± 10 nm screening filter which will read through microtiter plates. D. General Instructions Components of kit must be refrigerated when not in use. Kit is intended for one time use only; do not reuse wells containing test portion, reagents, or wash solution. Include single positive and negative control antigens with each group of tests. All controls must function properly for test to be valid. Use data record sheet to identify location of each test suspension. Use separate pipets for each test suspension and kit reagent to avoid cross-contamination. If plastic troughs are used to dispense conjugate and substrate, ensure that they are always kept separate. Components in kits are intended for use as integral unit. Do not mix components of different batch numbers. E. Preparation of Test Suspension ( a ) Pre-enrichment .—Pre-enrich product in noninhibitory broth to initiate growth of Salmonella spp. Methods used may vary with product and should be performed as indicated in 967.26A ( see 17.9.02), or in Bacteriological Analytical Manual , current edition, AOAC INTERNATIONAL, Gaithersburg, MD, USA, Chapter 5, section C. ( b ) Selective enrichment .—Transfer 1 mL incubated pre-enrichment mixtures to selenite cystine broth and 1 mL to tetrathionate broth as in 967.26B ( a ) ( see 17.9.02). For all foods other than raw foods or foods with a high microbial load, incubate 6–8 h at 35°C. Selective enrichments of raw foods or foods with a high microbial load must be incubated 16–20 h at 35 ° –37°C. ( c ) Post-enrichment .—Remove selective broths from incubation and mix by hand or with Vortex mixer. Remove 1 mL from tetrathionate tube and transfer to 10mL tube of sterileMbrothwhich has been warmed to 35 ° –37°C. Also remove 1 mL from selenite cystine tube and transfer to separate tube of M broth. For all foods other than raw foods or foods with a high microbial load, incubate Mbroth tubes 16–20 h and return selective enrichment broth tubes to 35 ° –37°C incubator and incubate for additional 16–18 h. For raw foods or foods with a highmicrobial load, incubateMbroth tubes 6 h at 35 ° –37°C and return selective enrichment broth tubes to 35 ° –37°C and incubate for additional 6 h at 35 ° –37°C.

( d ) Preparation of test suspension for EIA analysis .—Remove M broth tubes from incubation and mix tubes by hand or Vortex mixer. Combine equal aliquots of up to 1.0 mL from each M broth tube in clean screw-cap tube and heat in boiling water bath or in flowing steam 15 min. Refrigerate (2 ° –8°C) remaining M broth and tetrathionate and selenite cystine tubes from E ( c ) for culture confirmation of any EIA positive tests. Cool heated M broths to 25 ° –37°C prior to analysis by EIA. F. Enzyme Immunoassay ( 1 ) Following reagents must be prepared prior to commencing assay: ( a ) Prepare working strength wash solution by diluting contents of one vial of wash solution concentrate to 2 L with distilled or deionized H 2 O into reagent bottle. Plastic squeeze bottle is ideal for washing trays manually. ( b ) Prepare reconstituted positive control by transferring 3 mL control diluent to vial of lyophilized positive control antigen; mix thoroughly. Use the remaining control diluent as negative control. ( c ) Prepare reconstituted conjugate by adding vial of conjugate diluent to vial of lyophilized conjugate. Let conjugate rehydrate at room temperature. Gently mix reconstituted conjugate. ( d ) Prepare reconstituted substrate by adding vial of substrate diluent to lyophilized substrate. Be sure substrate has dissolved and mixture is at room temperature prior to use. Reconstituted substrate will appear pale green. ( e ) Use stop solution as received. No reconstitution is required. ( 2 ) Secure desired number of test (Removawell) strips in tray, allowing one well per food test suspension plus 2 wells for controls. Press wells firmly into place. Remove sealing film from top of wells to be used. Transfer 0.2 mL of each heated M broth test portion to single well. Transfer 0.2 mL aliquot of reconstituted negative control into one well and 0.2 mL aliquot of reconstituted positive control into another well. Record test suspension position on test sample record sheet provided. ( Note : Be sure that numbered tag at end of each test strip has been removed.) ( 3 ) Cover tray and incubate at 35 ° –37°C for 30 min in standard laboratory incubator. Tray must be covered to prevent evaporation. ( 4 ) After incubation, wash plate by hand, using plastic squeeze bottle containing working strength wash solution or use automatic washer charged with working strength wash solution as follows: ( a ) Quickly invert tray, emptying its contents into container. ( b ) Remove any residual liquid by firmly tapping tray face-down

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