AOAC ERP MICRO AUGUST 2018

17.9.13

Table 989.14A. 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)

AOAC Official Method 989.14 Salmonella in Foods Colorimetric Polyclonal Immunoassay Screening Method with Selenite Cystine Broth and Tetrathionate Broth (3M ä TECRA ä Salmonella Visual Immunoassay)

95 % Confidence range (approximately)

Results

Percent

Agreement a

First Action 1989 Final Action 1991 Modification 1997 Final Action 1999

96.8

95.4–98.2

False negative (BAM) b False negative (EIA) c

1.6 1.4

0.5–2.7

( e ) Conjugate .—Two vials (lyophilized). Contains ca 150 ng anti- Salmonella antibodies (from sheep) conjugated to horseradish peroxidase, 0.00686 g Na 2 B 4 O 7 , 0.12 g Dextran T10, 0.06 g hydrolyzed gelatin, 0.0024 g CaCl 2 , and 120 ng thimerosal. Reconstituted conjugate is stable for 28 days when stored at 2 ° –8°C. ( f ) Conjugate diluent .—Two vials (13.5 mL/vial). Contains 0.42 g Na 2 B 4 O 7 , 0.193 g NaCl, 0.22 g hydrolyzed gelatin, and 0.0022 g thimerosal in H 2 O. ( g ) Substrate .—One vial (lyophilized). Contains 0.011 g 2,2 ¢ -azino-di(3-ethylbenzthiazoline sulfonate) and 0.123 g NaH 2 PO 4 . Reconstituted substrate is stable 2 months when stored at 2 ° –8°C. ( h ) Substrate diluent .—One vial (22 mL/vial). Contains 0.116 g citric acid, 0.0011 g H 2 O 2 , and 0.0185 g NaOH in H 2 O. ( i ) Stop solution .—One vial (6 mL/vial). Vial contains 0.15 g NaF in H 2 O. Caution : Avoid contact with skin. If contact occurs, wash area with H 2 O. ( j ) Wash solution concentrate .—One vial (25 mL/vial). Contains 1.45 gTris, 7.03 gNaCl, 1.0 gTween, and 0.0025 g thimerosal inH 2 O. ( k ) Package insert . ( l ) Data record sheet . ( m ) Color comparator card .—For visual interpretation of positive and negative tests. ( n ) M broth .—5.0 g yeast extract, 12.5 g tryptone, 2.0 g D -mannose, 5.0 g sodium citrate, 5.0 g NaCl, 5.0 g K 2 HPO 4 , 0.14 g MnCl 2 , 0.8 g MgSO 4 , 0.04 g FeSO 4 , and 0.75 g Tween 80. Suspend ingredients in 1 L H 2 O and heat to boiling for 1–2 min. Dispense 10 mL portions into 16 ´ 125 mm screw-cap test tubes. Cap tubes loosely and autoclave 15 min at 121°C. Tighten caps securely for storage. Final pH should be 7.0 ± 0.2. ( o ) Diagnostic reagents .—Necessary for culture confirmation of presumptive positive EIA tests; see 967.25B ( see 17.9.01). C. Apparatus ( a ) Incubator .—35 ° –37°C. ( b ) Multipipets .—Delivering 20 and 250 m L volumes. ( c ) Water bath .—Maintaining 100°C. Autoclave set at 100°C is acceptable alternative, as are generators of flowing steam. ( d ) Plastic squeeze bottle .—500 mL, for dispensing wash solution. Automatic washer may be used. ( e ) Plastic film wrap or sealable plastic container .—To cover wells during incubation. 0.4–2.4 a This rate reflects number of test portions read identically between AOAC/BAM [ Bacteriological Analytical Manual, current edition, AOAC INTERNATIONAL, Gaithersburg, MD, USA] culture method and EIA. b This rate reflects number of test portions found to be positive by EIA but negative by AOAC/BAM culture method. c This rate reflects number of test portions found to be positive by AOAC/BAM culture method but negative by EIA.

[Method is screening procedure for presence of Salmonella in all foods; it is not a confirmatory test because polyclonal antibodies used in test may cross-react with small percentage of non- Salmonella . Enrichment broths and M broths from test suspensions positive by enzyme immunoassay (EIA) method must be streaked on selective media as in 967.26B ( see 17.9.02) and typical or suspicious colonies must be identified as in 967.26C ( see 17.9.02), 967.27 ( see 17.9.03), and 967.28 ( see 17.9.07).] Determination of positive result may be performed ( 1 ) visually by aid of color comparator card where positive result is valid when negative and positive controls match those described on card, or ( 2 ) instrumentally, using filter photometer, where positive result is valid only when negative and positive controls possess acceptable absorbance readings. See Tables 989.14A – C for the results of the interlaboratory study supporting acceptance of the method. A. Principle Detection of Salmonella antigens is based on EIA using highly purified antibodies prepared from antigens unique to Salmonella . Polyclonal antibodies to Salmonella antigen are absorbed onto internal surface of 96-well microtiter tray. Test suspension to be assayed is placed into a well of a tray. If Salmonella antigens are present in test suspension, they will attach to specific antibody absorbed on well. All other material in test suspensions is washed away. Conjugate is added and will bind to Salmonella antigens if they are attached to absorbed antibody on surface of well. Wells are washed to remove unbound conjugate, and enzyme substrate is added. Dark blue-green color indicates presence of Salmonella antigen in test portion. B. Reagents Items ( a )–( m ) are available as 3M TECRA Salmonella Visual Immunoassay from 3M Microbiology, 13 Rodborough Rd, Frenchs Forest, NSW2086, Australia; www.tecra.net; and 3MMicrobiology, 3M Center, Bldg 275-5W-05, St. Paul, MN 55144-1000, USA; www.3M.com/microbiology. Substitutions must be pretested for equivalency. ( a ) Antibody absorbed strips .—Microplate well strips, coated with polyclonal antibodies to Salmonella 96 wells. Store wells at 2 ° –8°C when not in use. ( b ) Tray .—Sufficient to secure individual wells or strips. ( c ) Control antigen .—Positive control (lyophilized). Purified Salmonella antigen, which reacts with antibodies to Salmonella , one vial. Reconstituted control antigen is stable 2 months when stored at 2 ° –8°C. ( d ) Controls diluent. —One vial (6 mL/vial). Contains 0.006 g Tris [tris(hydroxymethyl)aminomethane], 0.044 g NaCl, 0.0025 g Tween 20 (polyoxyethylene 20 sorbitan monolaurate), and 0.005 g thimerosal in H 2 O. Controls diluent also serves as negative control.

ã 2010 AOAC INTERNATIONAL