For over 100 years, Salmonella, one of the most frequently

Transcription

1 Bir et al.: Journal of AOAC International Vol. 96, No. 6, FOOD BIOLOGICAL CONTAMINANTS Evaluation of 3M Moleular Detetion Assay (MDA) Salmonella for the Detetion of Salmonella in Selete Foos: Collaorative Stuy Patrik Bir, Kiel Fisher, Megan Boyle, Travis Huffman, M. Joseph Benzinger, Jr, Paige Beinghaus, Jonathan Flannery, Erin Crowley, James Agin, an Davi Goins Q Laoratories, In., 1400 Harrison Ave, Cininnati, OH DeAnn Benesh 1 an John Davi 3M Foo Safety Department, 3M Center, Blg 260-6B-01, St. Paul, MN Collaorators: D. Awa, M. Banu, K. Blanhar, D. Boso, R. Brooks, D. Clark Jr, H. Dammann, J. Dyszel, V. Gill, M. Greenwell, C. Gwinn, M. Horan, J. Jurgens, M. Kelly, D. Lewis, S. Lue, J. Marhent, W. MMahon, I. Mello, S. Montez, S. Moosekian, A. Morey, K. Newman, M. Oltman, M. Ontieros, K. Rajkowski, J. Ruel, B. Stawik, L. Thompson, M. Vross The 3M Moleular Detetion Assay (MDA) Salmonella is use with the 3M Moleular Detetion System for the etetion of Salmonella spp. in foo, foo-relate, an environmental samples after enrihment. The assay utilizes loopmeiate isothermal amplifiation to rapily amplify Salmonella target DNA with high speifiity an sensitivity, omine with ioluminesene to etet the amplifiation. The 3M MDA Salmonella metho was ompare using an unpaire stuy esign in a multilaoratory ollaorative stuy to the U.S. Department of Agriulture/Foo Safety an Inspetion Servie-Miroiology Laoratory Guieook (USDA/FSIS-MLG 4.05), Isolation an Ientifiation of Salmonella from Meat, Poultry, Pasteurize Egg an Catfish Prouts for raw groun eef an the U.S. Foo an Drug Aministration/Bateriologial Analytial Manual (FDA/BAM) Chapter 5 Salmonella referene metho for wet og foo following the urrent AOAC guielines. A total of 20 laoratories partiipate. For the 3M MDA Salmonella metho, raw groun eef was analyze using 25 g test portions, an wet og foo was analyze using 375 g test portions. For the referene methos, 25 g test portions of eah matrix were analyze. Eah matrix was artifiially ontaminate with Salmonella at three inoulation levels: an uninoulate ontrol level (0 CFU/test portion), a low inoulum level (0.2 2 CFU/test portion), an a high inoulum level (2 5 CFU/test portion). In this stuy, 1512 unpaire repliate samples were analyze. Statistial analysis was onute aoring to the proaility of etetion (POD). For the low-level raw groun eef test portions, the following LPOD (ifferene etween the POD of the referene an aniate metho) Sumitte for puliation June 28, Corresponing author s [email protected] Appenixes are availale on the J. AOAC Int. wesite, pulisher.ingentaonnet.om/ontent/aoa/jaoa DOI: /jaoaint values with 95% onfiene intervals were otaine: 0.01 ( 0.14, +0.12). For the low-level wet og foo test portions, the following LPOD with 95% onfiene intervals were otaine: 0.04 ( 0.16, +0.09). No signifiant ifferenes were oserve in the numer of positive samples etete y the 3M MDA Salmonella metho versus either the USDA/FSIS-MLG or FDA/BAM methos. For over 100 years, Salmonella, one of the most frequently reporte auses of fooorne outreaks, has een known to ause fooorne illness in humans (1). The aterium has een impliate in outreaks from a variety of foos inluing raw animal prouts, suh as meat, poultry, eggs, airy prouts, seafoo, an some fruits an vegetales (2). In orer to reue outreaks of Salmonellosis, a omprehensive farm-to-fork approah is neee. The etetion of Salmonella an often e very time-onsuming an expensive, as the presene of the miroorganism in foo usually oes not affet the taste, smell, or appearane (3). The 3M Moleular Detetion Assay (MDA) Salmonella metho, in onjuntion with 3M Buffere Peptone Water ISO (BPW ISO; 4), uses a omination of loopmeiate isothermal DNA amplifiation an ioluminesene etetion to etet Salmonella in enrihe foo, fee, an environmental samples. The 3M MDA Salmonella metho allows for next-ay etetion of Salmonella speies. After h of enrihment using prewarme (37 ± 1 C) 3M BPW ISO meium, Salmonella etetion is performe y the 3M MDA Salmonella metho. Presumptive positive results are reporte in real time; negative results are isplaye after ompletion of the assay. Prior to the ollaorative stuy, the 3M MDA Salmonella metho was ertifie as a Performane Teste Metho (PTM) following the AOAC guielines for harmonize PTM stuies (5). The aim of the PTM stuy was to emonstrate that the 3M MDA Salmonella metho oul etet Salmonella in selete foos as laime y the manufaturer. For the 3M MDA Salmonella evaluation, six matries were analyze: raw groun eef (25 g), proesse reae hiken (325 g), liqui egg (100 g), shrimp (25 g), fresh spinah (25 g), an wet og foo (375 g). All other

2 2 Bir et al.: Journal of AOAC International Vol. 96, No. 6, 2013 PTM parameters (inlusivity, exlusivity, ruggeness, staility, an lot-to-lot variaility) teste in the PTM stuies satisfie the performane requirements for PTM approval. The metho was aware PTM ertifiation numer on Marh 30, The aim of this ollaorative stuy was to ompare the 3M MDA Salmonella metho to the U.S. Department of Agriulture (USDA) Foo Safety an Inspetion Servie (FSIS)-Miroiology Laoratory Guieook (MLG) 4.05 (6) for raw groun eef an the U.S. Foo an Drug Aministration (FDA) Bateriologial Analytial Manual (BAM) Chapter 5 (7) metho for wet og foo. Collaorative Stuy Stuy Design For this ollaorative stuy, two matries, raw groun eef (80% lean) an wet og foo (anne eef hunks), were analyze. The matries were otaine from loal retailers an sreene for the asene of Salmonella y preparing one ulk sample an analyzing five sample repliates (25 g) y the appropriate referene metho. The sreening iniate an asene of the target organism. The raw groun eef was artifiially ontaminate with Salmonella Ohio Sequene Types (STS) 81 an the wet og foo with Salmonella Poona National Colletion of Type Cultures (NCTC) There were two inoulation levels for eah matrix: a high inoulation level of approximately 2 5 CFU/test portion an a low inoulation level of approximately CFU/test portion. A set of uninoulate ontrol test portions was also inlue for eah matrix at 0 CFU/test portion. Twelve repliate samples from eah of the three ontamination levels of prout were analyze. Two sets of samples (72 total) were sent to eah laoratory for analysis y the 3M MDA Salmonella metho an either the USDA/FSIS-MLG (raw groun eef) or FDA/BAM (wet pet foo) referene metho ue to ifferent sample enrihments for the aniate metho an the referene methos. For oth matries, ollaorators were sent an aitional 30 g test portion an instrute to onut a total aeroi plate ount (APC) following the FDA/BAM Chapter 3 on the ay samples were reeive to etermine the total aeroi miroial loa. A etaile ollaorative stuy paket outlining all neessary information relate to the stuy inluing meia preparation, metho-speifi test portion preparation, an oumentation of results was sent to eah ollaorating laoratory prior to the initiation of the stuy. Preparation of Inoula an Test Portions The Salmonella ultures use in this evaluation were propagate in 10 ml of Brain Heart Infusion roth from a Q Laoratories frozen stok ulture hel at 70 C. The roth was inuate for h at 35 ± 1 C. Appropriate ilutions were prepare ase on previously estalishe growth urves for oth low an high inoulation levels, resulting in frational positive outomes for at least one level. For oth test portion sizes, a ulk lot of eah matrix was inoulate with a liqui inoulum an mixe thoroughly y han-kneaing to ensure an even istriution of miroorganisms. The matries were inoulate on the ay of shipment so that all test portions woul e hel for 96 h efore testing was initiate. For analysis of the raw groun eef, the ulk lot of test material was ivie into 30 g portions for shipment to the ollaorators. For analysis of the wet og foo, 25 g of inoulate test prout was mixe with 350 g of uninoulate test prout for shipment to the ollaorators for analysis y the 3M MDA Salmonella metho. For analysis y the referene metho, ollaoratoreeive 30 g portions. To etermine the level of Salmonella spp. in the matries, a five-tue most proale numer (MPN) was onute y the oorinating laoratory on the ay of initiation of analysis using the FDA/BAM Chapter 5 referene metho for wet pet foo or the USDA/FSIS-MLG 4.05 referene metho for raw groun eef. From oth the high an low inoulate levels, five 100 g test portions, the referene metho test portions, an five 10 g test portions were analyze using the appropriate referene metho enrihment roth. The MPN an 95% onfiene intervals were alulate from the high, low, an uninoulate levels using the MPN Calulator ( LCFMPNCalulator.exe; 8). Confirmation of the samples was onute aoring to either the USDA/FSIS-MLG 4.05 or FDA/BAM Chapter 5 referene metho, epenent on the matrix. Test Portion Distriution All samples were laele with a ranomize, lin-oe three-igit numer affixe to the sample ontainer. Test portions were shippe on a Thursay via overnight elivery aoring to the Category B Dangerous Goos shipment regulations set forth y the International Air Transport Assoiation. All samples were pake with ol paks to target a temperature of <7 C uring shipment. Upon reeipt, samples were hel y the ollaorating laoratory at refrigerate temperature (3 5 C) until the following Monay, when analysis was initiate. In aition to eah of the test portions an the total plate ount repliate, ollaorators also reeive a test portion for eah matrix laele as temperature ontrol. Partiipants were instrute to reor the temperature of this portion upon reeipt of the shipment, oument the results on the Sample Reeipt Confirmation form provie, an fax to the Stuy Diretor. Aitional shipments of raw groun eef test portions were mae y the sponsoring laoratory when aerrant results were oserve. Further investigation of the results iniate that eah partiipating ollaorator etete the presene of the target analyte in the uninoulate ontrol samples sent in the first shipment. In eah ase, the same speies was reporte for the ontrol samples, whih may have een ue to ross-ontamination. As a result, new test portions of raw groun eef were shippe an analyze y eah of the ollaorating laoratories. Test Portion Analysis Collaorators followe the appropriate preparation an analysis protool aoring to the metho for eah matrix. For oth matries, eah ollaorator reeive 72 test portions of eah foo prout (12 high, 12 low, an 12 ontrols for eah metho). For the analysis of the raw groun eef test portions y the 3M MDA Salmonella metho, a 25 g portion was enrihe with 225 ml of prewarme (37 ± 1 C) 3M BPW

3 Bir et al.: Journal of AOAC International Vol. 96, No. 6, ISO, homogenize for 2 min an inuate for 18 h at 37 ±1 C. For the wet og foo test portions analyze y the 3M MDA Salmonella metho, a 375 g portion was enrihe with 3375 ml prewarme (37 ± 1 C) 3M BPW ISO, homogenize for 2 min an inuate for 18 h at 37 ± 1 C. Following enrihment, samples were assaye y the 3M MDA Salmonella metho an onfirme following the stanar referene metho. Both test portion sizes analyze y the 3M MDA Salmonella metho were ompare to samples (25 g) analyze using either the USDA/FSIS-MLG or FDA/BAM referene metho in an unpaire stuy esign. All positive test portions were iohemially onfirme y the API 20E iohemial test, AOAC Offiial Metho , or y the VITEK 2 GN ientifiation test, AOAC Offiial Metho Serologial testing was also performe. Statistial Analysis Eah ollaorating laoratory reore results for the referene metho an the 3M MDA Salmonella metho on the ata sheets provie. The ata sheets were sumitte to the Stuy Diretor at the en of eah week of testing for analysis. The results of eah test portion for eah sample were ompile y the Stuy Diretor an the qualitative 3M MDA Salmonella results were ompare to the referene metho for statistial analysis. Data for eah test portion size were analyze using the proaility of etetion (POD; 9). If the onfiene interval of a LPOD i not ontain zero, then that woul iniate a statistially signifiant ifferene etween the aniate metho an the referene metho at the 5% onfiene level (9). AOAC Offiial Metho Salmonella in Selete Foos 3M Moleular Detetion Assay (MDA) Salmonella Metho First Ation 2013 [Appliale to etetion of Salmonella in raw groun eef (25 g), proesse reae hiken (325 g), liqui egg (100 g), shrimp (25 g), fresh spinah (25 g), an wet og foo (375 g)]. See Tales A an B for a summary of results of the inter-laoratory stuy. See Appenix Tales A an B for etaile results of the interlaoratory stuy. A. Priniple The 3M Moleular Detetion Assay (MDA) Salmonella metho is intene for use with the 3M Moleular Detetion System for the rapi an speifi etetion of Salmonella spp. in foo, fee, an environmental samples after enrihment. After enrihment in prewarme 3M Buffere Peptone Water ISO (3M BPW ISO) meium, the 3M MDA Salmonella test utilizes loopmeiate isothermal amplifiation to rapily amplify Salmonella target DNA with high speifiity an sensitivity, omine with ioluminesene to etet the amplifiation. Presumptive positive results are reporte in real time; negative results are isplaye after the assay is omplete. B. Apparatus an Reagents Items () (g) are availale as the 3M MDA Salmonella kit from 3M Foo Safety (St. Paul, MN). (a) 3M Moleular Detetion System. Availale from 3M Foo Safety. () 3M MDA Salmonella reagent tues. 12 strips of eight tues. () Lysis solution (LS) tues. 12 strips of eight tues. () Extra aps. 12 strips of eight aps. (e) Negative ontrol (NC). One vial (2 ml). (f) Reagent ontrol (RC). Eight reagent tues. (g) Quik start guie. (h) 3M Moleular Detetion Spee Loaer Tray. Availale from 3M Foo Safety. (i) 3M Moleular Detetion Chill Blok Tray an Chill Blok Insert. Availale from 3M Foo Safety. (j) 3M Moleular Detetion Heat Blok Insert. Availale from 3M Foo Safety. (k) 3M Moleular Detetion Cap/Deap Tool for reagent tues. Availale from 3M Foo Safety. (l) 3M Moleular Detetion Cap/Deap Tool for lysis tues. Availale from 3M Foo Safety. (m) Empty lysis tue rak. Availale from 3M Foo Safety. (n) Empty reagent tue rak. Availale from 3M Foo Safety. (o) 3M BPW ISO. Availale from 3M Foo Safety. Formulation equivalent to ISO 6579:2002 Annex B (4). (p) Disposale pipet. Capale of 20 µl. (q) Multihannel (eight-hannel) pipet. Capale of 20 µl. (r) Sterile filter tip pipet tips. Capale of 20 µl. (s) Filter stomaher ags. Sewar Laoratory Systems In., Bohemia, NY, or equivalent. (t) Stomaher. Sewar Laoratory Systems In. or equivalent. (u) Thermometer. Calirate range to inlue 100 ± 1 C. (v) Dry oule lok heater unit or water ath. Capale of maintaining 100 ± 1 C. (w) Inuators. Capale of maintaining 37 ± 1 C. (x) Freezer. Capale of maintaining 10 to 20 C, for storing the 3M Moleular Detetion Chill Blok Tray. (y) Refrigerator. Capale of maintaining 2 8 C, for storing the 3M MDA. (z) Computer. Compatile with the 3M Moleular Detetion Instrument. C. General Instrutions (a) Store the 3M MDA Salmonella kit at 2 8 C. Do not freeze. Keep kit away from light uring storage. After opening the kit, hek that the foil pouh is unamage. If the pouh is amage, o not use. After opening, unuse reagent tues shoul always e store in the resealale pouh with the esiant insie to maintain staility of the lyophilize reagents. Store reseale pouhes at 2 8 C for no longer than 60 ays. Do not use 3M MDA Salmonella past the expiration ate. () The 3M Moleular Detetion Instrument is intene for use with samples that have unergone heat treatment uring the assay lysis step, whih is esigne to estroy organisms present in the sample. Samples that have not een properly heat-treate uring the assay lysis step may e onsiere a potential

4 4 Bir et al.: Journal of AOAC International Vol. 96, No. 6, 2013 Tale A. POD summary of raw groun eef (25 g) results for the 3M MDA Salmonella metho a Inoulation level Uninoulate Low High Caniate presumptive positive/total No. of samples analyze 1/120 69/ /120 Caniate presumptive (CP) POD 0.01 (0.00, +0.05) 0.58 (+0.48, +0.67) 1.00 (+0.97, +1.00) 0.09 (+0.08, +0.17) 0.51 (+0.45, +0.52) 0.00 (0.00, +0.18) 0.00 (0.00, +0.04) 0.00 (0.00, +0.14) 0.00 (0.00, +0.18) 0.09 (+0.08, +0.10) 0.51 (+0.45, +0.52) 0.00 (0.00, +0.24) Caniate onfirme positive/total No. of samples analyze 0/120 67/ /120 Caniate onfirme (CC) POD 0.00 (0.00, +0.03) 0.56 (+0.47, +0.65) 1.00 (+0.97, +1.00) 0.00 (0.00, +0.17) 0.51 (+0.45, +0.52) 0.00 (0.00, +0.18) 0.00 (0.00, +0.17) 0.00 (0.00, +0.11) 0.00 (0.00, +0.18) 0.00 (0.00, +0.24) 0.51 (+0.46, +0.52) 0.00 (0.00, +0.24) Positive referene samples/total No. of samples analyze 0/120 68/ /120 Referene POD 0.00 (0.00, +0.03) 0.57 (+0.48, +0.66) 0.99 (+0.95, +1.00) 0.00 (0.00, +0.17) 0.50 (+0.45, +0.52) 0.09 (+0.08, +0.17) 0.00 (0.00, +0.17) 0.00 (0.00, +0.18) 0.00 (0.00, +0.04) 0.00 (0.00, +0.24) 0.51 (+0.45, +0.52) 0.09 (+0.08, 0.11) LPOD (Caniate veferene) 0.00 ( 0.03, +0.03) 0.01 ( 0.14, +0.12) 0.01 ( 0.02, +0.05) LPOD (CP vs CC) 0.01 ( 0.02, +0.05) 0.02 ( 0.11, +0.15) 0.00 ( 0.03, +0.03) a Results inlue 95% onfiene intervals. Repeataility SD. Among-laoratory SD. Reprouiility SD. iohazar an shoul not e inserte into the 3M Moleular Detetion Instrument. () Follow all instrutions arefully. Failure to o so may lea to inaurate results. () After use, the enrihment meium an the 3M MDA Salmonella tues an potentially ontain pathogeni materials. When testing is omplete, follow urrent inustry stanars for the isposal of ontaminate waste. Consult the Material Safety Data Sheet for aitional information an loal regulations for isposal. Perioially eontaminate laoratory enhes an equipment (pipets, ap/eap tools, et.) with a 1 5% (v/v in water) househol leah solution or DNA removal solution. D. Sample Enrihment Prewarm 3M BPW ISO enrihment meium to 37 ± 1 C. Aseptially omine the enrihment meium an sample following the outline in Tale C. For all meat an highly partiulate samples, the use of filter ags ieommene. Homogenize thoroughly for 2 min. Inuate at 37 ± 1 C. E. Preparation of the 3M Moleular Detetion Spee Loaer Tray Wet a loth or paper towel with a 1 5% (v/v in water) househol leah solution an wipe the 3M Moleular Detetion Spee Loaer Tray. Rinse the tray with water. Use a isposale towel to wipe the tray ry. Ensure the 3M Moleular Detetion Spee Loaer Tray iy efore use. F. Preparation of the 3M Moleular Detetion Chill Blok Insert Before using the 3M Moleular Detetion Chill Blok Insert, ensure it has een store on the 3M Moleular Detetion Chill Blok Tray in the freezer ( 10 to 20 C) for a minimum of 2 h efore use. When removing the 3M Moleular Detetion Chill Blok Insert from the freezer for use, remove it an the 3M Moleular Detetion Chill Blok Tray together. Use the insert an tray within 20 min. G. Preparation of the 3M Moleular Detetion Heat Blok Insert Plae the 3M Moleular Detetion Heat Blok Insert in a ry oule lok heater unit. Turn on the ry lok heater unit an set the temperature to allow the 3M Moleular Detetion Heat Blok Insert to reah an maintain a temperature of 100 ± 1 C. Note: Depening on the heater unit, allow approximately min for the 3M Moleular Detetion Heat Blok Insert to reah temperature. Using a alirate thermometer, verify that the 3M Moleular Detetion Heat Blok Insert is at 100 ± 1 C.

5 Bir et al.: Journal of AOAC International Vol. 96, No. 6, Tale B. POD Summary of wet pet foo (375 g) results for the 3M MDA Salmonella metho a Inoulation level Uninoulate Low High Caniate presumptive positive/total No. of samples analyze 1/132 65/ /132 Caniate presumptive (CP) POD 0.01 (0.00, +0.04) 0.49 (+0.40, +0.58) 0.99 (+0.96, +1.00) 0.09 (+0.08, +0.16) 0.51 (+0.46, +0.52) 0.09 (+0.08, +0.16) 0.00 (0.00, +0.04) 0.00 (0.00, +0.14) 0.00 (0.00, +0.04) 0.09 (+0.08, +0.10) 0.51 (+0.46, +0.52) 0.09 (+0.08, +0.10) Caniate onfirme positive/total No. of samples analyze 0/132 65/ /132 Caniate onfirme (CC) POD 0.00 (0.00, +0.03) 0.49 (+0.40, +0.58) 0.99 (+0.96, +1.00) 0.00 (0.00, +0.17) 0.51 (+0.46, +0.52) 0.09 (+0.08, +0.16) 0.00 (0.00, +0.17) 0.00 (0.00, +0.14) 0.00 (0.00, +0.04) 0.00 (0.00, +0.23) 0.51 (+0.46, +0.52) 0.09 (+0.08, +0.10) Positive referene samples/total No. of samples analyze 0/132 70/ /132 Referene POD 0.00 (0.00, +0.03) 0.53 (+0.44, +0.62) 1.00 (+0.97, +1.00) 0.00 (0.00, +0.17) 0.52 (+0.46, +0.52) 0.00 (0.00, +0.17) 0.00 (0.00, +0.17) 0.00 (0.00, +0.09) 0.00 (0.00, +0.17) 0.00 (0.00, +0.23) 0.52 (+0.47, +0.52) 0.00 (0.00, +0.23) LPOD (Caniate veferene) 0.00 ( 0.03, +0.03) 0.04 ( 0.16, +0.09) 0.01 ( 0.04, +0.02) LPOD (CP vs CC) 0.01 ( 0.02, +0.05) 0.00 ( 0.13, +0.13) 0.00 ( 0.03, +0.03) a Results inlue 95% onfiene intervals. Repeataility SD. Among-laoratory SD. Reprouiility SD. H. Preparation of the 3M Moleular Detetion Instrument Launh the 3M Moleular Detetion Software an log in. Turn on the 3M Moleular Detetion Instrument. Create or eit a run with ata for eah sample. Refer to the 3M Moleular Detetion System User Manual for etails. Note: The 3M Moleular Detetion Instrument must reah an maintain a temperature of 60 C efore a run an e starte. This heating step takes approximately 20 min an is iniate y an orange light on the instrument s status ar. When the instrument ieay to start a run, the status ar will turn green. I. Lysis Allow the LS tues to warm up to room temperature y setting the rak on the laoratory enh for 2 h. Alternatives to equilirate the LS tues to room temperature are to inuate the LS tues in a 37 ± 1 C inuator for 1 h or at room temperature overnight (16 18 h). Remove the enrihment roth from the inuator an gently agitate the ontents. One LS tue is require for eah sample an the NC sample. LS tue strips an e ut to the esire numer. Selet the numer of iniviual LS tues or eight-tue strips neee. Plae the LS tues in an empty rak. To avoi ross-ontamination, eap strip at a time an use a new pipet tip for eah transfer step. Transfer the enrihe samples to LS tues as esrie elow: Note: Transfer eah enrihe sample into iniviual LS tue first. Transfer the NC last. Use the 3M Moleular Detetion Cap/Deap Tool-Lysis to eap one LS tue strip one strip at a time. Set the tool with ap attahe asie on a lean surfae. Transfer 20 µl of sample into an LS tue. Repeat transfer until eah iniviual sample has een ae to a orresponing LS tue in the strip. Use the 3M Moleular Detetion Cap/Deap Tool-Lysis to reap the LS tue strip. Use the roune sie of the tool to apply pressure in a ak-an-forth motion to ensure that the ap is tightly applie. Repeat as neee for the numer of samples to e teste. When all samples have een transferre, transfer 20 µl of NC into a LS tue. Use the 3M Moleular Detetion Cap/ Deap Tool-Lysis tool to reap the LS tue. Cover the rak of LS tues with the rak li an firmly invert three to five times Tale C Sample enrihment protools Sample matrix Sample size, g Enrihment roth volume, ml Enrihment time, h Raw groun eef (27% fat) Raw shrimp Bagge spinah Pasteurize liqui whole egg Cooke reae hiken Wet pet foo (og eef uts in gravy, anne)

6 6 Bir et al.: Journal of AOAC International Vol. 96, No. 6, 2013 Figure A. Transfer of enrihe sample to Lysis Solution tue. Figure B. Sample Lysis. to mix. Suspension has to flow freely insie the tue. See Figure A. Verify that the temperature of the 3M Moleular Detetion Heat Blok Insert is at 100 ± 1 C. Plae the rak of LS tues in the 3M Moleular Detetion Heat Blok Insert an heat for 15 ± 1 min. An alternative to using ry heat for the lysis step is to use a water ath at 100 ±1 C. Ensure that suffiient water is use to over up to the liqui level in the LS tues. Plae the rak of LS tues in the water ath at 100 ± 1 C an heat for 15 ± 1 min. Samples that have not een properly heat-treate uring the assay lysis step may e onsiere a potential iohazar an shoul not e inserte into the 3M Moleular Detetion Instrument. Remove the rak of LS tues from the heating lok an allow to ool in the 3M Moleular Detetion Chill Blok Insert for 10 ± 1 min. Remove the rak li uring inuation on the 3M Moleular Detetion Chill Blok Insert. The LS solution may freeze when proessing less than 48 LS tues. Freezing of the LS solution will not affet your test. If freezing is oserve, allow the LS tues to thaw for 5 min efore mixing. Remove the rak of LS tues from the 3M Moleular Detetion Chill Blok Insert/3M Moleular Detetion Chill Blok Tray system. Replae the li on the rak of LS tues an firmly invert three to five times to mix. Suspension has to flow freely insie the tue. Firmly tap the lysis tueak on the laoratory enh three to five times. Plae the rak on the laoratory enh. Let it sit unisture for at least 5 min to allow the resin to settle. Do not mix or istur the resin at the ottom of the tue. See Figure B. J. Amplifiation One reagent tue iequire for eah sample an the NC. Reagent tue strips an e ut to esire tue numer. Selet the numer of iniviual reagent tues or eight-tue strips neee. Plae reagent tues in an empty rak. Avoi isturing the reagent pellets from the ottom of the tues. Selet one RC tue an plae in rak. To avoi rossontamination, eap one reagent tues strip at a time an use a new pipet tip for eah transfer step. Transfer lysate to reagent tues an RC tue as follows: Transfer eah sample lysate into iniviual reagent tues first followe y the NC. Hyrate the RC tue last. Warning: Care must e taken when pipetting LS, as arry-over of the resin may interfere with amplifiation. (1) Use the 3M Moleular Detetion Cap/Deap Tool-Reagent to eap the reagent tues one strip at a time. Disar ap. (2) Transfer 20 µl of sample lysate from the upper portion of the flui in the LS tue into orresponing reagent tue. Dispense at an angle to avoi isturing the pellets. Mix y gently pipetting up an own five times. (3) Repeat until iniviual sample lysate has een ae to a orresponing reagent tue in the strip. (4) Cover the reagent tues with the provie extra ap an use the roune sie of the 3M Moleular Detetion Cap/Deap Tool-Reagent to apply pressure in a ak-an-forth motion, ensuring that the ap is tightly applie. Repeat steps (1) to (4) as neee for the numer of samples to e teste. When all sample lysates have een transferre, repeat steps (1) to (4) to transfer 20 µl of NC lysate into a reagent tue. Transfer 20 µl of NC lysate into a RC tue. Dispense at an angle to avoi isturing the pellets. Mix y gently pipetting up an own five times. Loa appe tues into a lean an eontaminate 3M Moleular Detetion Spee Loaer Tray. Close an lath the 3M Moleular Detetion Spee Loaer Tray li. See Figure C. Review an onfirm the onfigure run in the 3M Moleular Detetion Software. Clik the start utton in the software an selet instrument for use. The selete instrument s li automatially opens. Plae the 3M Moleular Detetion Spee Loaer Tray into the 3M Moleular Detetion Instrument an lose the li to start the assay. Results are provie within 75 min, although positives may e etete sooner. After the assay is omplete, remove the 3M Moleular Detetion Spee Loaer Tray from the 3M Moleular Detetion Figure C. Transfer of lysate to reagent tue.

7 Bir et al.: Journal of AOAC International Vol. 96, No. 6, Instrument an ispose of the tues y soaking in a 1 5% (v/v in water) househol leah solution for 1 h an away from the assay preparation area. Notie: To minimize the risk of false positives ue to ross-ontamination, never open reagent tues ontaining amplifie DNA. This inluec, reagent, an matrix ontrol tues. Always ispose of seale reagent tues y soaking in a 1 5% (v/v in water) househol leah solution for 1 h away from the assay preparation area. K. Results an Interpretation An algorithm interprets the light output urve resulting from the etetion of the nulei ai amplifiation. Results are analyze automatially y the software an are olor-oe ase on the result. A positive or negative result is etermine y analysis of a numer of unique urve parameters. Presumptive positive results are reporte in real time; negative an inspet results will e isplaye after the run is omplete. Presumptive positive results shoul e onfirme using your preferre metho or as speifie y the FDA/BAM ( BateriologialAnalytialManualBAM/um htm) or the USDA/FSIS-MLG ( MLG_4_05.pf; 6, 7), starting from the 3M BPW ISO, followe y seonary enrihment, plating, an onfirmation of isolates using appropriate iohemial an serologial methos. Note: Even a negative sample will not give a zero reaing as the system an 3M MDA Salmonella amplifiation reagents have a akgroun relative light unit. In the rare event of any unusual light output, the algorithm laels this as inspet. 3M reommens the user to repeat the assay for any inspet samples. If the result ontinues to e inspet, proee to onfirmation test using your preferre metho or as speifie y loal regulations. Results In this ollaorative stuy, the 3M MDA Salmonella metho was ompare to the to the USDA/FSIS-MLG 4.05 referene metho for raw groun eef an to the FDA/BAM, Chapter 5 referene metho for wet og foo. A total of 20 laoratories throughout the Unite States partiipate in this stuy, with 14 laoratories sumitting ata for the raw groun eef an 16 laoratories sumitting ata for the wet og foo, as presente in Tale 1. Eah laoratory analyze 36 test portions for eah metho: 12 inoulate with a high level of Salmonella, 12 inoulate with a low level of Salmonella, an 12 uninoulate ontrols. For eah matrix, the atual level of Salmonella was etermine y MPN etermination on the ay of initiation of analysis y the oorinating laoratory. The iniviual laoratory an sample results are presente in Tales 2 an 3. Tales A an B summarize the interlaoratory results for all foos teste, inluing POD statistial analysis (10). The results of the ollaorating laoratories APC analysis for eah matrix are presente in Tale C of the Appenix. Raw Groun Beef (25 g Test Portions) Raw groun eef test portions were inoulate at a low an high level an were analyze (Tale 2) for the etetion of Tale 1. Partiipation of eah ollaorating laoratory a La Raw groun eef (25 g test portions) Wet og foo (375 g test portions) 1 Y Y 2 Y Y 3 N Y 4 N Y 5 N Y 6 N Y 7 N Y 8 N Y 9 Y Y 10 Y Y 11 Y Y 12 Y Y 13 Y Y 14 Y Y 15 Y Y 16 Y Y 17 Y N 18 Y N 19 Y N 20 Y N a Y = Collaorator analyze the foo type; N = ollaorator i not analyze the foo type. Data otaine from aitional shipment of raw groun eef. Initial shipment of raw groun eef was not use for evaluation purposes an therefore the ata has not een presente. Results were not use in statistial analysis ue to laoratory error, or uninoulate ontrol test portions were onfirme as Salmonella. Salmonella spp. Uninoulate ontrols were inlue in eah analysis. The results presente for the raw groun eef were from a seon shipment of test portions to the ollaorating laoratories. The initial shipment of raw groun eef test portions sent to ollaorators was isovere to ontain ontamination of the target analyte in the uninoulate ontrol samples for eah laoratory an therefore no ata have een presente. Fourteen laoratories partiipate in the retest analysis of this matrix an the results of 10 laoratories were inlue in the statistial analysis. For the retest of the raw groun eef, laoratories 12, 16, 18, an 19 etete the presene of Salmonella spp. in either the aniate or referene metho ontrol repliates. Beause of the potential for error, results from these laoratories were exlue from the statistial analysis. The MPN levels otaine for this test portion, with 95% onfiene intervals, were 0.81 CFU/test portion (+0.62, +1.04) for the low level an 4.68 CFU/test portion (+3.22, +6.80) for the high level. For the high level, 120 out of 120 test portions were reporte as presumptive positive y the 3M MDA Salmonella metho with all test portions onfirming positive. For the low level, 67 out of 120 test portions were reporte as presumptive positive y the 3M MDA Salmonella metho with 65 test portions onfirming positive. For the uninoulate ontrols, 1 out of 120 samples proue a presumptive positive result y the

8 8 Bir et al.: Journal of AOAC International Vol. 96, No. 6, 2013 Tale 2. Iniviual ollaorator results for raw groun eef (25 g test portions) a High-level test portionow-level test portions Uninoulate test portions La M MDA Salmonella NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 4 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 6 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 7 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 8 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA USDA/FSIS-MLG NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 4 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 6 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 7 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 8 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA a + = Salmonella spp. were etete in samples; =Salmonella spp. were not etete in sample; NA = laoratory i not partiipate in this matrix, or results were not reeive. Sample results were otaine from the seon shipment of raw groun eef test portions. Sample was presumptive positive on 3M MDA Salmonella, ut onfirme negative, iniating a false-positive result. Results were not use in statistial analysis ue to laoratory error.

9 Bir et al.: Journal of AOAC International Vol. 96, No. 6, Tale 3. Iniviual ollaorator results for wet og foo (375 g test portions) a High-level test portionow-level test portions Uninoulate test portions La M MDA Salmonella NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 18 NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 19 NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 20 NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA FDA/BAM NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 18 NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 19 NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 20 NA NA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA a + = Salmonella spp. were etete in samples; =Salmonella spp. were not etete in sample; NA = laoratory i not partiipate in this matrix or results were not reeive. Results were not use in statistial analysis ue to laoratory error. Sample was presumptive positive on 3M MDA Salmonella, ut onfirme negative, iniating a false-positive result.

10 10 Bir et al.: Journal of AOAC International Vol. 96, No. 6, M MDA Salmonella metho with all test portions onfirming negative. For test portions analyze y the USDA/FSIS-MLG Metho, 119 out of 120 high inoulum an 68 out of 120 low inoulum test portions onfirme positive. For the uninoulate ontrols, 0 out of 120 test portions onfirme positive. For the low-level inoulum, a LPOD C value of 0.01 with 95% onfiene intervals of ( 0.14, +0.13) were otaine etween the 3M MDA Salmonella metho an the USDA/FSIS-MLG metho. The onfiene intervals otaine for LPOD C iniate no signifiant ifferene etween the two methos. A LPOD CP value of 0.02 with 95% onfiene intervals of ( 0.11, +0.15) was otaine etween presumptive an onfirme 3M MDA Salmonella results. The onfiene intervals otaine for LPOD CP iniate no signifiant ifferene etween the presumptive an onfirme results using either onfirmation proess. For the high-level inoulum, a LPOD C value of 0.01 with 95% onfiene intervals of ( 0.02, +0.05) was otaine etween the 3M MDA Salmonella metho an the USDA/FSIS-MLG metho. The onfiene intervals otaine for LPOD C iniate no signifiant ifferene etween the two methos. A LPOD CP value of 0.00 with 95% onfiene intervals of ( 0.03, +0.03) was otaine etween presumptive an onfirme 3M MDA Salmonella results. The onfiene intervals otaine for LPOD CP iniate no signifiant ifferene etween the presumptive an onfirme results. Detaile results of the POD statistial analysis are presente in Tale A an Figures 1A an B of the Appenix. Wet Dog Foo (375 g Test Portions) Wet og foo test portions were inoulate at a low an high level an were analyze (Tale 3) for the etetion of Salmonella spp. Uninoulate ontrols were inlue in eah analysis. Sixteen laoratories partiipate in the analysis of this matrix an the results of 11 laoratories were inlue in the statistial analysis. Laoratories 4, 5, 10, an 16 etete the presene of Salmonella spp. in either the aniate or referene metho ontrol repliates. Beause of the potential for error, results from these laoratories were exlue from the statistial analysis. Laoratory 12 i not sumit results ue to ross-ontamination of sample enrihments aeporte y the analyst. The MPN levels otaine for this test portion, with 95% onfiene intervals, were 0.72 CFU/test portion (+0.57, +0.90) for the low level an 5.34 CFU/test portion (+3.46, +8.24) for the high level. For the high level, 131 out of 132 test portions were reporte as presumptive positive y the 3M MDA Salmonella metho with all test portions onfirming positive. For the low level, 65 out of 132 test portions were reporte as presumptive positive y the 3M MDA Salmonella metho with all test portions onfirming positive. For the uninoulate ontrols, 1 out of 132 samples proue a presumptive positive result y the 3M MDA Salmonella metho with all test portions onfirming negative. For test portions analyze y the FDA/BAM metho, 132 out of 132 high inoulum an 70 out of 132 low inoulum test portions onfirme positive. For the uninoulate ontrols, 0 out of 132 test portions onfirme positive. For the low-level inoulum, a LPOD C value of 0.04 with 95% onfiene intervals of ( 0.16, +0.09) was otaine etween the 3M MDA Salmonella metho an the FDA/BAM metho. The onfiene intervals otaine for LPOD C iniate no signifiant ifferene etween the two methos. A LPOD CP value of 0.00 with 95% onfiene intervals of ( 0.13, +0.13) was otaine etween presumptive an onfirme 3M MDA Salmonella results. The onfiene intervals otaine for LPOD CP iniate no signifiant ifferene etween the presumptive an onfirme results using either onfirmation proess. For the high-level inoulum, a LPOD C value of 0.01 with 95% onfiene intervals of ( 0.04, +0.02) was otaine etween the 3M MDA Salmonella metho an the FDA/BAM metho. The onfiene intervals otaine for LPOD C iniate no signifiant ifferene etween the two methos. A LPOD CP value of 0.00 with 95% onfiene intervals of ( 0.03, +0.03) was otaine etween presumptive an onfirme 3M MDA Salmonella results. The onfiene intervals otaine for LPOD CP iniate no signifiant ifferene etween the presumptive an onfirme results. Detaile results of the POD statistial analysis are presente in Tale B an Figures 2A an B of the Appenix. Disussion For this ollaorative stuy, samples were analyze at oth 25 an 375 g test portions aequire y the urrent AOAC Guielines (5), whih require methos with more than one sample preparation or enrihment sheme to analyze one matrix per proeure. No negative feeak was provie y the ollaorating laoratories in regar to the performane of the aniate metho. Several ollaorating laoratories expresse questions in regar to the AOAC stuy esign of the ollaorative stuy; others expresse onern with analyzing 375 g test portions. The onern with hanling the larger test portions may have ontriute to errors oserve uring testing that resulte in ata not use in the statistial analysis. During testing, four ifferent laoratories etete the presene of Salmonella spp. in seven raw groun eef uninoulate ontrol test portions. Aitionally, four ifferent laoratories etete the presene of Salmonella spp. in 15 wet pet foo uninoulate ontrol test portions. Due to eteting positive samples in the ontrol test portions, the ata provie y these laoratories were not inlue uring the statistial analysis. A root ause investigation to etermine the soure of ontamination yiele the following possiilities: Due to the high numer of samples analyze, inluing test portions inoulate at a high inoulum level, ontamination may have ourre uring the transfer of enrihe samples into the seonary seletive enrihments or uring the streaking of the referene agar plates. For the wet pet foo, ase on feeak from the ollaorators, issues with storage uring the inuation of the larger test portion sizes may have le to ross-ontamination of the primary enrihments. Base on the fat that uninoulate ontrol test portions were pakage 1 ay prior to the inoulate test portions, ontamination uring test portion preparation at the oorinating laoratory is not elieve to e the ause of the positive ontrol samples. During the analysis of oth the raw groun eef an wet pet foo, some laoratories proue false-positive results with the aniate metho. The 3M Moleular Detetion Assay is intene for use in a laoratory environment y professionals

11 Bir et al.: Journal of AOAC International Vol. 96, No. 6, traine in laoratory tehnique. Cross-ontamination of sampleesulting in false-positive results may our if areful moleular tehniques are not followe. To reue the risk of ross-ontamination, 3M reommens the use of sterile, aerosol arrier (filtere) moleular iology grae pipet tips. A new pipet tip shoul e use for eah sample transfer, an the user may hoose to a an intermeiate transfer step in orer to avoi pipet ontamination, i.e., eah enrihe sample an e transferre into a sterile tue efore proeeing to the lysis step. Disrepant results may e otaine if eviations from the metho our. Use of alirate pipettors an thermometers is ritial to ensure that orret volumes of samples, espeially when hyrating the reagent tues, an appropriate temperatures are utilize. It ieommene that userea an eome familiar with the 3M MDA Salmonella prout instrutions an follow them arefully. For either matrix, the ollaorative stuy faile to show a statistially signifiant ifferene etween the aniate metho an the referene metho using the POD moel when the aforementione four laoratories were remove from onsieration. Reommenations It ieommene that the 3M MDA Salmonella metho e aopte Offiial First Ation for the etetion of Salmonella in selete foos, inluing raw groun eef (25 g), proesse reae hiken (325 g), liqui egg (100 g), shrimp (25 g), fresh spinah (25 g), an wet og foo (375 g). Aknowlegments We exten our sinere thanks to the following ollaorators for their eiate partiipation in this stuy: Joanne Ruel, Cherney Miroiologial Servies, Lt, Green Bay, WI Jessia Dyszel an Mathew Vross, Rihter International, Columus, OH Vikas Gill, U.S. FDA Center for Foo Safety an Applie Nutrition, College Park, MD Bra Stawik an Keith Blanhar, Miroa Laoratories, In., Warrenale, PA Mark Horan an Delano Lewis, Miroa Laoratories, In., Baltimore, MD Inaue Mello an Maria Ontieros, Mars Petare, US, Kansas City, MO Joene Jurgens an Leslie Thompson, Aegis, North Sioux City, SD Davi Boso, Foo Safety Net Servies, Fresno, CA Amit Morey an Sergio Montez, Foo Safety Net Servies, San Antonio, TX Kyle Newman, Venture Laoratories, In., Lexington, KY Mary Banu an Matt Oltman, Chestnut Laoratory, Springfiel, MO Roert Brooks, ATC Miroiology, LLC, North Little Rok, AR Christine Gwinn an Sott Moosekian, Covane Laoratories, In., Battle Creek, MI Joey Marhent, Gulf Coast Seafoo Laoratory, FDA, Dauphin Islan, AL Kathleen T. Rajkowski, USDA Agriultural Researh Servies, Eastern Regional Researh Center, Foo Safety an Tehnologies Initiative, Glensie, PA Shaunti Lue, The National Foo La, Livermore, CA Hono Dammann an Dorn Clark Jr, Marshfiel Foo Safety, Marshfiel, WI Weny MMahon an Deena Awa, Silliker, In., Crete, IL Mihelle Kelly an Megan Greenwell, Q Laoratories In., Cininnati, OH Referenes (1) Centers for Disease Control an Prevention (Otoer 26, 2011) < (aesse Novemer 6, 2012) (2) Hammak, Thomas (2011) Salmonella speies in Ba Bug Book Fooorne Pathogeni Miroorganisms an Natural Toxins, 2n E., U.S. Foo an Drug Aministration, College Park, MD (3) U.S. Department of Agriulture, Foo Safety an Inspetion Servie (May 2007) < etail/a_i/334/relate/1> (aesse Novemer 6, 2012) (4) International Organization for Stanarization (2002) ISO 6579: Miroiology of Foo an Animal Feeing Stuffs-Horizontal Metho for the Detetion of Salmonella spp., 4th E., Geneva, Switzerlan (5) Brunelle, S., LaBue, R., Nelson, M., & Wehling, P. (2012) AOAC INTERNATIONAL Methos Committee Guielines for Valiation of Miroiologial Methos for Foo an Environmental Surfaes, AOAC INTERNATIONAL, Gaithersurg, MD, Appenix X (6) U.S. Department of Agriulture, Foo Safety an Inspetion Servie, Miroiology Laoratory Guieook, Revision 4.05 (2001) Isolation an Ientifiation of Salmonella from Meat, Poultry, Pasteurize Egg, an Catfish Prouts, Washington, DC. (aesse July 2012) (7) Anrews, W.H., & Hammak, T. (Feruary 2011) FDA- Bateriologial Analytial Manual, Chapter 5 Salmonella. LaoratoryMethos/ BateriologialAnalytialManualBAM/um htm (aesse July 2012) (8) Least Cost Formulations, Lt (2011) MPN Calulator-Version (aesse Novemer 2012) (9) Wehling, P., LaBue, R., Brunelle, S., & Nelson, M. (2011) J. AOAC Int. 94, (10) Least Cost Formulations, Lt (2011) AOAC Binary Data Interlaoratory Stuy Workook. (aesse Novemer 2012)