AOAC ERP MICRO AUGUST 2018

Table 998.09D. Performance parameters

Incidence of false negatives among total positive suspensions, % d

Incidence of false positives among total negative suspensions, % f

Sensitivity, % e

Specificity, % g

Agreement, % b

Level j

Product

ULTIMA Culture

ULTIMA Culture

ULTIMA Culture

ULTIMA Culture

Milk powder

U

100 100 100 100 100 100 100 95 100

0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

100 100

100 100

L

100 100

100 100

H U

Peanut butter

100 100

100 100

L

100 100 100

100 100 100

H

Raw poultry

1 2 3

100 100

100 100

5.9

94.1 100

0

100

100

Note : Footnotes a–i refer to Tables 998.09A–D . a Most probable number of colony forming units per gram of food. b Rate reflects the number of confirmed determinations that were equivalent between the 3M TECRA and culture methods. c c 2 is defined by McNemar as (|a – b| – 1) 2 /(a + b) where a = suspensions positive by 3M TECRA and negative by culture method and b = suspensions negative by 3M TECRA and positive by culture method. A c 2 value greater than 3.84 indicates significance at P < 0.05. d Incidence of false negatives is 100 – sensitivity rate. e Sensitivity rate is defined as 100 times the total number of analyzed positive suspensions among “known” positive suspensions divided by total number of “known” suspensions, where “known” positive is defined as suspensions confirmed positive by the reference method. f Incidence of false positives is 100 – specificity rate. g Specificity rate is defined as 100 times the total number of analyzed negative suspensions among “known” negative suspensions divided by the total number of “known” negative suspensions, where “known” negative is defined as suspensions confirmed negative by the reference method and negative controls. h — = Statistical analysis not applicable. i r = Reader only; v = visual only. j U = Uninoculated; L = low; H = high; 1 = very low level of natural contamination (MPN <0.03); 2 = low level of natural contamination (MPN 0.03); 3 = high level of natural contamination (MPN 1.5).

INTERNATIONAL, Gaithersburg, MD 20877, USA, Chapter 5, section C. Incubate pre-enrichment broths at 36 ° ± 1 ° C. ( b ) Selective enrichment.— Transfer 0.1 mL incubated pre-enrichment mixture to Rappaport-Vassiliadis R10 broth (9.9 mL). Incubate 18–24 h at 42 ° ± 1 ° C. ( c ) A d d i t i o n o f s a m p l e a d d i t i v e . —R e m o v e Rappaport-Vassiliadis R10 from incubation and mix by hand or by Vortex mixer. Place 25 m L additive, B ( l ), into a tube, add 1 mL incubated Rappaport-Vassiliadis R10 to this and mix in a Vortex mixer. Retain remaining Rappaport-Vassiliadis R10 broth in refrigerator for later confirmation if necessary. ( d ) Preparation for EIA analysis .—Heat Rappaport-Vassiliadis R10 + additive in boiling H 2 O bath or in flowing steam 15 min. Cool heat-treated broths to 25 ° –37 ° C prior to analysis by EIA, G . G. Enzyme Immunoassay ( 1 ) Prepare following reagents prior to beginning assay: ( a ) Prepare working-strength wash solution by diluting contents of 1 vial of wash solution concentrate to 2 L with distilled or deionized H 2 O in 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 substratewill appear pale green. ( e ) Use stop solution as received. No reconstitution is required. ( 2 ) Secure desired number of test strips (Removawell) in tray, allowing 1 well per test plus 2 wells for controls. Press wells firmly into place. Remove sealing film from top of wells to be used. Transfer 0.2 mL aliquots of the heat-treated broths from each test suspension to a single well. Transfer 0.2mLaliquots of reconstituted positive and negative contols into individual wells. Record test sample position on record sheet provided. ( 3 ) Cover tray with plastic film and incubate at 36 ° ± 1 ° 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 on paper towel several times. ( c ) Completely fill eachwell withworking strengthwash solution. ( d ) Repeat ( a )–( c ) 2 more times.

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