Evaluation of 3M Molecular Detection Assay (MDA) Salmonella for
Contents
1. 4 HF He RE 19 E da a a ER A ASE a a A e 1 gt 20 4 He Ke Ke o a results were not received Sample results were obtained from the second shipment of raw ground beef test portions Sample was presumptive positive on 3M MDA Salmonella but confirmed negative indicating a false positive result Results were not used in statistical analysis due to laboratory error Salmonella spp were detected in samples Salmonella spp were not detected in sample NA laboratory did not participate in this matrix or BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 96 NO 6 2013 9 Table 3 Individual collaborator results for wet dog food 375 g test portions High level test portions Low level test portions Uninoculated test portions Lab 1 2 3 4 567 89 101112 123 4 5 6 7 8 9 10 1112 1 2 3 4 5 6 7 8 9 10 11 12 3M MDA Salmonella 1 4 4 4 4 HF o o o He HH ER gt ee 2 t ot 4 4 HF FH o 4 4 o 4 4 0 He ee ee ee Ke KK Ke 3 4 4 4 4 o o Htt 4 4 o A A A A A ce nono E A A A A E e e G S e l o a 6 4 4 oooh o o 4 4 4 4 4 o 7 ot 4 4 4 o 4 4 o 4 4 o ee ee Ke eK eK Ke 8 4 FH 42. 4 4 HO HE Hee He ee Ke eK Ke 9 E E E E E EE E E 10 TEE E A A AE l 1 4 4 4 4 4 12 NA NA NA NA NANANANANA 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 13 Eo E E E EG 4 2 1S Se eee Se Se eS Ss 14 4 4 o tHe ee ee ee ee Ke Ke Ke Ke 15 4 4 4 Je 2 E A de AA A E e ES a ear O eH 17 NA NA NA NA NANANANANA 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 18 NA NA NA NA NANANANANA 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 19 NA NA NA NA NANANANANA 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 20 NA NA NA NA NANANANANA 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 FDA BAM 1 C 4 4 4 4 4 o o 2 E Es E ELA A OE A E E ee er el 3 C 4 4 o 4 re 4 gt 4 4 o 3 5P EOC E 4 4 4 o o o o o o o 6 HEO 4 4 4 4 4 4 o o o 7 HEO 4 4 4 4 4 4 4 o o o 8 E E AA o A e E tof ee
3. Place the 3M Molecular Detection Heat Block Insert in a dry double block heater unit Turn on the dry block heater unit and set the temperature to allow the 3M Molecular Detection Heat Block Insert to reach and maintain a temperature of 100 1 C Note Depending on the heater unit allow approximately 30 50 min for the 3M Molecular Detection Heat Block Insert to reach temperature Using a calibrated thermometer verify that the 3M Molecular Detection Heat Block Insert is at 100 1 C Table 2013 09B POD Summary of wet pet food 375 g results for the 3M MDA Salmonella method Candidate presumptive positive total No of samples analyzed Candidate presumptive CP POD s s1 sp Candidate confirmed positive total No of samples analyzed Candidate confirmed CC POD s sis SR Positive reference samples total No of samples analyzed Reference POD sp Si Sa dLPOD Candidate vs Reference dLPOD CP vs CC Inoculation level BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 96 NO 6 2013 Uninoculated 1 132 0 01 0 00 0 04 0 09 0 08 0 16 0 00 0 00 0 04 0 09 0 08 0 10 0 132 0 00 0 00 0 03 0 00 0 00 0 17 0 00 0 00 0 17 0 00 0 00 0 23 0 132 0 00 0 00 0 03 0 00 0 00 0 17 0 00 0 00 0 17 0 00 0 00 0 23 0 00 0 03 0 03 0 01 0 02 0 05 Low 65 132 0 49 0 40 0 58 0 51 0 46 0 52 0 00 0 00 0 14 0 51 0 46 0 52 65
4. lean and wet dog food canned beef chunks were analyzed The matrices were obtained from local retailers and screened for the absence of Salmonella by preparing one bulk sample and analyzing five sample replicates 25 g by the appropriate reference method The screening indicated an absence of the target organism The raw ground beef was artificially contaminated with Salmonella Ohio Sequence Types STS 81 and the wet dog food with Salmonella Poona National Collection of Type Cultures NCTC 4840 There were two inoculation levels for each matrix a high inoculation level of approximately 2 5 CFU test portion and a low inoculation level of approximately 0 2 2 CFU test portion A set of uninoculated control test portions was also included for each matrix at 0 CFU test portion Twelve replicate samples from each of the three contamination levels of product were analyzed Two sets of samples 72 total were sent to each laboratory for analysis by the 3M MDA Salmonella method and either the USDA FSIS MLG raw ground beef or FDA BAM wet pet food reference method due to different sample enrichments for the candidate method and the reference methods For both matrices collaborators were sent an additional 30 g test portion and instructed to conduct a total aerobic plate count APC following the FDA BAM Chapter 3 on the day samples were received to determine the total aerobic microbial load A detailed collaborative study packet outlining all ne
5. 132 0 49 0 40 0 58 0 51 0 46 0 52 0 00 0 00 0 14 0 51 0 46 0 52 70 132 0 53 0 44 0 62 0 52 0 46 0 52 0 00 0 00 0 09 0 52 0 47 0 52 0 04 0 16 0 09 0 00 0 13 0 13 High 131 132 0 99 0 96 1 00 0 09 0 08 0 16 0 00 0 00 0 04 0 09 0 08 0 10 131 132 0 99 0 96 1 00 0 09 0 08 0 16 0 00 0 00 0 04 0 09 0 08 0 10 132 132 1 00 0 97 1 00 0 00 0 00 0 17 0 00 0 00 0 17 0 00 0 00 0 23 0 01 0 04 0 02 0 00 0 03 0 03 Results include 95 confidence intervals gt Repeatability SD Among laboratory SD Y Reproducibility SD H Preparation of the 3M Molecular Detection Instrument Launch the 3M Molecular Detection Software and log in Turn on the 3M Molecular Detection Instrument Create or edit a run with data for each sample Refer to the 3M Molecular Detection System User Manual for details Note The 3M Molecular Detection Instrument must reach and maintain a temperature of 60 C before a run can be started This heating step takes approximately 20 min and is indicated by an orange light on the instrument s status bar When the instrument is ready to start a run the status bar will turn green I Lysis Allow the LS tubes to warm up to room temperature by setting the rack on the laboratory bench for 2 h Alternatives to equilibrate the LS tubes to room temperature are to incubate the LS tubes in a 37
6. MLG 4 05 pdf 6 7 starting from the 3M BPW ISO followed by secondary enrichment plating and confirmation of isolates using appropriate biochemical and serological methods Note Even a negative sample will not give a zero reading as the system and 3M MDA Salmonella amplification reagents have a background relative light unit In the rare event of any unusual light output the algorithm labels this as inspect 3M recommends the user to repeat the assay for any inspect samples If the result continues to be inspect proceed to confirmation test using your preferred method or as specified by local regulations Results In this collaborative study the 3M MDA Salmonella method was compared to the to the USDA FSIS MLG 4 05 reference method for raw ground beef and to the FDA BAM Chapter 5 reference method for wet dog food A total of 20 laboratories throughout the United States participated in this study with 14 laboratories submitting data for the raw ground beef and 16 laboratories submitting data for the wet dog food as presented in Table 1 Each laboratory analyzed 36 test portions for each method 12 inoculated with a high level of Salmonella 12 inoculated with alow level of Salmonella and 12 uninoculated controls For each matrix the actual level of Salmonella was determined by MPN determination on the day of initiation of analysis by the coordinating laboratory The individual laboratory and sample results are presented
7. No 6 2013 3M MDA Salmonella method with all test portions confirming negative For test portions analyzed by the USDA FSIS MLG Method 119 out of 120 high inoculum and 68 out of 120 low inoculum test portions confirmed positive For the uninoculated controls 0 out of 120 test portions confirmed positive For the low level inoculum a dLPOD value of 0 01 with 95 confidence intervals of 0 14 0 13 were obtained between the 3M MDA Salmonella method and the USDA FSIS MLG method The confidence intervals obtained for dLPOD indicated no significant difference between the two methods A dLPOD p value of 0 02 with 95 confidence intervals of 0 11 0 15 was obtained between presumptive and confirmed 3M MDA Salmonella results The confidence intervals obtained for dLPODcp indicated no significant difference between the presumptive and confirmed results using either confirmation process For the high level inoculum a dLPOD value of 0 01 with 95 confidence intervals of 0 02 0 05 was obtained between the 3M MDA Salmonella method and the USDA FSIS MLG method The confidence intervals obtained for dLPOD indicated no significant difference between the two methods A dLPOD p value of 0 00 with 95 confidence intervals of 0 03 0 03 was obtained between presumptive and confirmed 3M MDA Salmonella results The confidence intervals obtained for dLPODcp indicated no significant difference between the presumptive and confirmed re
8. as temperature control Participants were instructed to record the temperature of this portion upon receipt of the shipment document the results on the Sample Receipt Confirmation form provided and fax to the Study Director Additional shipments of raw ground beef test portions were made by the sponsoring laboratory when aberrant results were observed Further investigation of the results indicated that each participating collaborator detected the presence of the target analyte in the uninoculated control samples sent in the first shipment In each case the same species was reported for the control samples which may have been due to cross contamination As a result new test portions of raw ground beef were shipped and analyzed by each of the collaborating laboratories Test Portion Analysis Collaborators followed the appropriate preparation and analysis protocol according to the method for each matrix For both matrices each collaborator received 72 test portions of each food product 12 high 12 low and 12 controls for each method For the analysis of the raw ground beef test portions by the 3M MDA Salmonella method a 25 g portion was enriched with 225 mL of prewarmed 37 1 C 3M BPW BIRD ET AL ISO homogenized for 2 min and incubated for 18 h at 37 41 C For the wet dog food test portions analyzed by the 3M MDA Salmonella method a 375 g portion was enriched with 3375 mL prewarmed 37 1 C 3M BPW ISO homogenize
9. er el 9 bo Eo E O E E E E O E E to oo 10 11 EC CE HE Eo E O E E 4 E os o o gt 12 NA NA NA NA NANANANANA 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 13 C 4 0 o 14 ECO o 4 15 ECO 16 t 17 NA NA NA NA NANANANANA NA NANA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA NA NA 18 NA NA NA NA NANANANANA 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 19 NA NA NA NA NANANANANA 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 20 NA NA NA NA NANANANANA 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 2 Salmonella spp were detected in samples Salmonella spp were not detected in sample NA laboratory did not participate in this matrix or results were not received gt Results were not used in statistical analysis due to laboratory error Sample was presumptive positive on 3M MDA Salmonella but confirmed negative indicating a false positive result 10 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 96
10. high inoculum level contamination may have occurred during the transfer of enriched samples into the secondary selective enrichments or during the streaking of the reference agar plates For the wet pet food based on feedback from the collaborators issues with storage during the incubation of the larger test portion sizes may have led to cross contamination of the primary enrichments Based on the fact that uninoculated control test portions were packaged 1 day prior to the inoculated test portions contamination during test portion preparation at the coordinating laboratory is not believed to be the cause of the positive control samples During the analysis of both the raw ground beef and wet pet food some laboratories produced false positive results with the candidate method The 3M Molecular Detection Assay is intended for use in a laboratory environment by professionals BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 96 NO 6 2013 11 trained in laboratory technique Cross contamination of samples resulting in false positive results may occur if careful molecular techniques are not followed To reduce the risk of cross contamination 3M recommends the use of sterile aerosol barrier filtered molecular biology grade pipet tips A new pipet tip should be used for each sample transfer and the user may choose to add an intermediate transfer step in order to avoid pipet contamination i e each enriched sample can be transferred into a st
11. was discovered to contain contamination of the target analyte in the uninoculated control samples for each laboratory and therefore no data have been presented Fourteen laboratories participated in the retest analysis of this matrix and the results of 10 laboratories were included in the statistical analysis For the retest of the raw ground beef laboratories 12 16 18 and 19 detected the presence of Salmonella spp in either the candidate or reference method control replicates Because of the potential for error results from these laboratories were excluded from the statistical analysis The MPN levels obtained for this test portion with 95 confidence intervals were 0 81 CFU test portion 0 62 1 04 for the low level and 4 68 CFU test portion 3 22 6 80 for the high level For the high level 120 out of 120 test portions were reported as presumptive positive by the 3M MDA Salmonella method with all test portions confirming positive For the low level 67 out of 120 test portions were reported as presumptive positive by the 3M MDA Salmonella method with 65 test portions confirming positive For the uninoculated controls 1 out of 120 samples produced a presumptive positive result by the 8 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 96 No 6 2013 Table 2 Individual collaborator results for raw ground beef 25 g test portions High level test portions Low level test portions Uninoculated test portions Lab 1 2 3 4 5 6 7
12. 1 C incubator for 1 h or at room temperature overnight 16 18 h Remove the enrichment broth from the incubator and gently agitate the contents One LS tube is required for each sample and the NC sample LS tube strips can be cut to the desired number Select the number of individual LS tubes or eight tube strips needed Place the LS tubes in an empty rack To avoid cross contamination decap strip at a time and use a new pipet tip for each transfer step Transfer the enriched samples to LS tubes as described below Note Transfer each enriched sample into individual LS tube first Transfer the NC last Use the 3M Molecular Detection Cap Decap Tool Lysis to decap one LS tube strip one strip at a time Set the tool with cap attached aside on a clean surface Transfer 20 uL of sample into an LS tube Repeat transfer until each individual sample has been added to a corresponding LS tube in the strip Use the 3M Molecular Detection Cap Decap Tool Lysis to recap the LS tube strip Use the rounded side of the tool to apply pressure in a back and forth motion to ensure that the cap is tightly applied Repeat as needed for the number of samples to be tested When all samples have been transferred transfer 20 uL of NC into a LS tube Use the 3M Molecular Detection Cap Decap Tool Lysis tool to recap the LS tube Cover the rack of LS tubes with the rack lid and firmly invert three to five times Table 2013 09C Sample enrichment protocols Sample s
13. 8 9 10 1112 123 45 6 7 8 9 10 11 12 1 2 3 45 6 7 8 9 10 11 12 3M MDA Salmonella 1 o E E E t r E eee 2 t t t EA A p 3 t 3 NA NA NA NA NA NA NA NA NANA NA NA NA NA NANA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA 4 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 5 NA NA NA NA NA NA NA NA NANA NA NA NA NA NANA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 6 NA NA NA NA NA NA NA NA NANA NA NA NA NA NANA NA NA NA NANA 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 NANA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NANANA NA NA 8 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 9 0 gt 10 4 ee ee ee ee Fe 11 E O o 44 o E E ae E A Rt 13 a a fe fe _ 14 E E E ERE EE ot HF o eK 15 t gt ee a se e 16 SR ae e o AE E BS AE EEE OS ek II Bk 17 HF 4 4 E E E E E E E TE E E E E E 0 Ec E da A AE E OE SS ee E ALS
14. BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 96 NO 6 2013 1 FOOD BIOLOGICAL CONTAMINANTS Evaluation of 3M Molecular Detection Assay MDA Salmonella for the Detection of Salmonella in Selected Foods Collaborative Study PATRICK BIRD KIEL FISHER MEGAN BOYLE TRAVIS HUFFMAN M JOSEPH BENZINGER JR PAIGE BEDINGHAUS JONATHAN FLANNERY ERIN CROWLEY JAMES AGIN and DAVID GOINS Q Laboratories Inc 1400 Harrison Ave Cincinnati OH 45214 DEANN BENESH and JOHN DAVID 3M Food Safety Department 3M Center Bldg 260 6B 01 St Paul MN 55144 Collaborators D Awad M Bandu K Blanchard D Bosco R Brooks D Clark Jr H Dammann J Dyszel V Gill M Greenwell C Gwinn M Horan J Jurgens M Kelly D Lewis S Luce J Marchent W McMahon I Mello S Montez S Moosekian A Morey K Newman M Oltman M Ontiberos K Rajkowski J Ruebl B Stawick L Thompson M Vross The 3M Molecular Detection Assay MDA Salmonella is used with the 3M Molecular Detection System for the detection of Salmonella spp in food food related and environmental samples after enrichment The assay utilizes loop mediated isothermal amplification to rapidly amplify Salmonella target DNA with high specificity and sensitivity combined with bioluminescence to detect the amplification The 3M MDA Salmonella method was compared using an unpaired study design in a multilaboratory collaborative study to the U S Department of Agri
15. OD Candidate vs Reference dLPOD CP vs CC 1 120 0 01 0 00 0 05 0 09 0 08 0 17 0 00 0 00 0 04 0 09 0 08 0 10 0 120 0 00 0 00 0 03 0 00 0 00 0 17 0 00 0 00 0 17 0 00 0 00 0 24 0 120 0 00 0 00 0 03 0 00 0 00 0 17 0 00 0 00 0 17 0 00 0 00 0 24 0 00 0 03 0 03 0 01 0 02 0 05 69 120 0 58 0 48 0 67 0 51 0 45 0 52 0 00 0 00 0 14 0 51 0 45 0 52 67 120 0 56 0 47 0 65 0 51 0 45 0 52 0 00 0 00 0 11 0 51 0 46 0 52 68 120 0 57 0 48 0 66 0 50 0 45 0 52 0 00 0 00 0 18 0 51 0 45 0 52 0 01 0 14 0 12 0 02 0 11 0 15 120 120 1 00 0 97 1 00 0 00 0 00 0 18 0 00 0 00 0 18 0 00 0 00 0 24 120 120 1 00 0 97 1 00 0 00 0 00 0 18 0 00 0 00 0 18 0 00 0 00 0 24 119 120 0 99 0 95 1 00 0 09 0 08 0 17 0 00 0 00 0 04 0 09 0 08 0 11 0 01 0 02 0 05 0 00 0 03 0 03 Results include 95 confidence intervals gt Repeatability SD Among laboratory SD d Reproducibility SD biohazard and should not be inserted into the 3M Molecular Detection Instrument c Follow all instructions carefully Failure to do so may lead to inaccurate results d After use the enrichment medium and the 3M MDA Salmonella tubes can potentially contain pathogenic materials When testing is complete follow current industry standards for the disposal of contaminate
16. askfsis custhelp com app answers detail a_id 334 related 1 gt accessed November 6 2012 4 International Organization for Standardization 2002 SO 6579 Microbiology of Food and Animal Feeding Stuffs Horizontal Method for the Detection of Salmonella spp 4th Ed Geneva Switzerland 5 Brunelle S LaBudde R Nelson M amp Wehling P 2012 AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces AOAC INTERNATIONAL Gaithersburg MD Appendix X 6 U S Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Revision 4 05 2001 Isolation and Identification of Salmonella from Meat Poultry Pasteurized Egg and Catfish Products Washington DC http www fsis usda gov PDF MLG_4 05 pdf accessed July 2012 7 Andrews W H amp Hammack T February 2011 FDA Bacteriological Analytical Manual Chapter 5 Salmonella http www fda gov Food ScienceResearch LaboratoryMethods BacteriologicalAnalyticalManualBAM ucm070149 htm accessed July 2012 8 Least Cost Formulations Ltd 2011 MPN Calculator Version 1 6 www lefltd com customer LCFMPNCalculator exe accessed November 2012 9 Wehling P LaBudde R Brunelle S amp Nelson M 2011 J AOAC Int 94 335 347 10 Least Cost Formulations Ltd 2011 AOAC Binary Data Interlaboratory Study Workbook http lcfitd com aoac aoac binary v2 2 xls access
17. ble number MPN was conducted by the coordinating laboratory on the day of initiation of analysis using the FDA BAM Chapter 5 reference method for wet pet food or the USDA FSIS MLG 4 05 reference method for raw ground beef From both the high and low inoculated levels five 100 g test portions the reference method test portions and five 10 g test portions were analyzed using the appropriate reference method enrichment broth The MPN and 95 confidence intervals were calculated from the high low and uninoculated levels using the MPN Calculator www lcfltd com customer LCFMPNCalculator exe 8 Confirmation of the samples was conducted according to either the USDA FSIS MLG 4 05 or FDA BAM Chapter 5 reference method dependent on the matrix Test Portion Distribution All samples were labeled with a randomized blind coded three digit number affixed to the sample container Test portions were shipped on a Thursday via overnight delivery according to the Category B Dangerous Goods shipment regulations set forth by the International Air Transport Association All samples were packed with cold packs to target a temperature of lt 7 C during shipment Upon receipt samples were held by the collaborating laboratory at refrigerated temperature 3 5 C until the following Monday when analysis was initiated In addition to each of the test portions and the total plate count replicate collaborators also received a test portion for each matrix labeled
18. cessary information related to the study including media preparation method specific test portion preparation and documentation of results was sent to each collaborating laboratory prior to the initiation of the study Preparation of Inocula and Test Portions The Salmonella cultures used in this evaluation were propagated in 10 mL of Brain Heart Infusion broth from a Q Laboratories frozen stock culture held at 70 C The broth was incubated for 18 24 h at 35 1 C Appropriate dilutions were prepared based on previously established growth curves for both low and high inoculation levels resulting in fractional positive outcomes for at least one level For both test portion sizes a bulk lot of each matrix was inoculated with a liquid inoculum and mixed thoroughly by hand kneading to ensure an even distribution of microorganisms The matrices were inoculated on the day of shipment so that all test portions would be held for 96 h before testing was initiated For analysis of the raw ground beef the bulk lot of test material was divided into 30 g portions for shipment to the collaborators For analysis of the wet dog food 25 g of inoculated test product was mixed with 350 g of uninoculated test product for shipment to the collaborators for analysis by the 3M MDA Salmonella method For analysis by the reference method collaborators received 30 g portions To determine the level of Salmonella spp in the matrices a five tube most proba
19. culture Food Safety and Inspection Service Microbiology Laboratory Guidebook USDA FSIS MLG 4 05 Isolation and Identification of Salmonella from Meat Poultry Pasteurized Egg and Catfish Products for raw ground beef and the U S Food and Drug Administration Bacteriological Analytical Manual FDA BAM Chapter 5 Salmonella reference method for wet dog food following the current AOAC guidelines A total of 20 laboratories participated For the 3M MDA Salmonella method raw ground beef was analyzed using 25 g test portions and wet dog food was analyzed using 375 g test portions For the reference methods 25 g test portions of each matrix were analyzed Each matrix was artificially contaminated with Salmonella at three inoculation levels an uninoculated control level 0 CFU test portion a low inoculum level 0 2 2 CFU test portion and a high inoculum level 2 5 CFU test portion In this study 1512 unpaired replicate samples were analyzed Statistical analysis was conducted according to the probability of detection POD For the low level raw ground beef test portions the following dLPOD difference between the POD of the reference and candidate method Submitted for publication June 28 2013 i Corresponding author s e mail dbenesh1 mmm com Appendixes are available on the J AOAC Int website http aoac publisher ingentaconnect com content aoac jaoac DOI 10 5740 jaoacint 13 227 values with 95 confidence intervals w
20. d for 2 min and incubated for 18 h at 37 1 C Following enrichment samples were assayed by the 3M MDA Salmonella method and confirmed following the standard reference method Both test portion sizes analyzed by the 3M MDA Salmonella method were compared to samples 25 g analyzed using either the USDA FSIS MLG or FDA BAM reference method in an unpaired study design All positive test portions were biochemically confirmed by the API 20E biochemical test AOAC Official Method 978 24 or by the VITEK 2 GN identification test AOAC Official Method 2011 17 Serological testing was also performed Statistical Analysis Each collaborating laboratory recorded results for the reference method and the 3M MDA Salmonella method on the data sheets provided The data sheets were submitted to the Study Director at the end of each week of testing for analysis The results of each test portion for each sample were compiled by the Study Director and the qualitative 3M MDA Salmonella results were compared to the reference method for statistical analysis Data for each test portion size were analyzed using the probability of detection POD 9 If the confidence interval of a dLPOD did not contain zero then that would indicate a statistically significant difference between the candidate method and the reference method at the 5 confidence level 9 AOAC Official Method 2013 09 Salmonella in Selected Foods 3M Molecular Detection Assay MDA Salmone
21. d waste Consult the Material Safety Data Sheet for additional information and local regulations for disposal Periodically decontaminate laboratory benches and equipment pipets cap decap tools etc with a 1 5 v v in water household bleach solution or DNA removal solution D Sample Enrichment Prewarm 3M BPW ISO enrichment medium to 37 1 C Aseptically combine the enrichment medium and sample following the outline in Table 2013 09C For all meat and highly particulate samples the use of filter bags is recommended Homogenize thoroughly for 2 min Incubate at 37 1 C E Preparation of the 3M Molecular Detection Speed Loader Tray Wet a cloth or paper towel with a 1 5 v v in water household bleach solution and wipe the 3M Molecular Detection Speed Loader Tray Rinse the tray with water Use a disposable towel to wipe the tray dry Ensure the 3M Molecular Detection Speed Loader Tray is dry before use F Preparation of the 3M Molecular Detection Chill Block Insert Before using the 3M Molecular Detection Chill Block Insert ensure it has been stored on the 3M Molecular Detection Chill Block Tray in the freezer 10 to 20 C for a minimum of 2 h before use When removing the 3M Molecular Detection Chill Block Insert from the freezer for use remove it and the 3M Molecular Detection Chill Block Tray together Use the insert and tray within 20 min G Preparation of the 3M Molecular Detection Heat Block Insert
22. e control NC One vial 2 mL f Reagent control RC Eight reagent tubes g Quick start guide h 3M Molecular Detection Speed Loader Tray Available from 3M Food Safety i 3M Molecular Detection Chill Block Tray and Chill Block Insert Available from 3M Food Safety j 3M Molecular Detection Heat Block Insert Available from 3M Food Safety k 3M Molecular Detection Cap Decap Tool for reagent tubes Available from 3M Food Safety D 3M Molecular Detection Cap Decap Tool for lysis tubes Available from 3M Food Safety m Empty lysis tube rack Available from 3M Food Safety n Empty reagent tube rack Available from 3M Food Safety 0 3M BPW ISO Available from 3M Food Safety Formulation equivalent to ISO 6579 2002 Annex B 4 p Disposable pipet Capable of 20 uL q Multichannel eight channel pipet Capable of 20 uL r Sterile filter tip pipet tips Capable of 20 uL s Filter stomacher bags Seward Laboratory Systems Inc Bohemia NY or equivalent t Stomacher Seward Laboratory Systems Inc or equivalent u Thermometer Calibrated range to include 100 1 C v Dry double block heater unit or water bath Capable of maintaining 100 1 C w Incubators Capable of maintaining 37 1 C x Freezer Capable of maintaining 10 to 20 C for storing the 3M Molecular Detection Chill Block Tray y Refrigerator Capable of maintaining 2 8 C for s
23. e provided within 75 min although positives may be detected sooner After the assay is complete remove the 3M Molecular Detection Speed Loader Tray from the 3M Molecular Detection Figure 2013 09C Transfer of lysate to reagent tube BIRD ET AL Instrument and dispose of the tubes by soaking in a 1 5 v v in water household bleach solution for 1 h and away from the assay preparation area Notice To minimize the risk of false positives due to cross contamination never open reagent tubes containing amplified DNA This includes RC reagent and matrix control tubes Always dispose of sealed reagent tubes by soaking in a 1 5 v v in water household bleach solution for 1 h away from the assay preparation area K Results and Interpretation An algorithm interprets the light output curve resulting from the detection of the nucleic acid amplification Results are analyzed automatically by the software and are color coded based on the result A positive or negative result is determined by analysis of a number of unique curve parameters Presumptive positive results are reported in real time negative and inspect results will be displayed after the run is completed Presumptive positive results should be confirmed using your preferred method or as specified by the FDA BAM http www fda gov Food ScienceResearch LaboratoryMethods BacteriologicalAnalyticalManualBAM ucm070149 htm or the USDA FSIS MLG http www fsis usda gov PDF
24. ed November 2012
25. ere obtained 0 01 0 14 0 12 For the low level wet dog food test portions the following dLPOD with 95 confidence intervals were obtained 0 04 0 16 0 09 No significant differences were observed in the number of positive samples detected by the 3M MDA Salmonella method versus either the USDA FSIS MLG or FDA BAM methods reported causes of foodborne outbreaks has been known to cause foodborne illness in humans 1 The bacterium has been implicated in outbreaks from a variety of foods including raw animal products such as meat poultry eggs dairy products seafood and some fruits and vegetables 2 In order to reduce outbreaks of Salmonellosis a comprehensive farm to fork approach is needed The detection of Salmonella can often be very time consuming and expensive as the presence of the microorganism in food usually does not affect the taste smell or appearance 3 The 3M Molecular Detection Assay MDA Salmonella method in conjunction with 3M Buffered Peptone Water ISO BPW ISO 4 uses a combination of loop mediated isothermal DNA amplification and bioluminescence detection to detect Salmonella in enriched food feed and environmental samples The 3M MDA Salmonella method allows for next day detection of Salmonella species After 18 24 h of enrichment using prewarmed 37 1 C 3M BPW ISO medium Salmonella detection is performed by the 3M MDA Salmonella method Presumptive positive results are reported i
26. erile tube before proceeding to the lysis step Discrepant results may be obtained if deviations from the method occur Use of calibrated pipettors and thermometers is critical to ensure that correct volumes of samples especially when hydrating the reagent tubes and appropriate temperatures are utilized It is recommended that users read and become familiar with the 3M MDA Salmonella product instructions and follow them carefully For either matrix the collaborative study failed to show a statistically significant difference between the candidate method and the reference method using the POD model when the aforementioned four laboratories were removed from consideration Recommendations It is recommended that the 3M MDA Salmonella method be adopted Official First Action for the detection of Salmonella in selected foods including raw ground beef 25 g processed breaded chicken 325 g liquid egg 100 g shrimp 25 g fresh spinach 25 g and wet dog food 375 g Acknowledgments We extend our sincere thanks to the following collaborators for their dedicated participation in this study Joanne Ruebl Cherney Microbiological Services Ltd Green Bay WI Jessica Dyszel and Mathew Vross Richter International Columbus OH Vikas Gill U S FDA Center for Food Safety and Applied Nutrition College Park MD Brad Stawick and Keith Blanchard Microbac Laboratories Inc Warrendale PA Mark Horan and Delando Lewis Microbac Laborat
27. es a a a a sa E e 20 t t AE t t ESE AER E H USDA FSIS MLG 1 t t He 2 t t AE Eo HH E E t t 4 l fF eee 3 NA NA NA NA NA NA NA NA NANA NA NA NA NA NANA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA 4 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 5 NA NA NA NA NA NA NA NA NANA NA NA NA NA NANA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NANA 6 NA NA NA NA NA NA NA NA NANA NA NA NA NA NANA NA NA NA NANA 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 NANA NA NA NA NANA 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 NANA NA NA NA NANA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 9 wo ok E t y E HH E E H H f eee 10 t 4 HF SF o 11 t 4 ee ee qo a ag e o 13 gt 14 t o t A o E E E E a F 4 15 4 4 HH 4 RE Ke 16 gt oo gt gt ee 17
28. esults of the POD statistical analysis are presented in Table 2013 09B and Figures 2A and B of the Appendix Discussion For this collaborative study samples were analyzed at both 25 and 375 g test portions as required by the current AOAC Guidelines 5 which require methods with more than one sample preparation or enrichment scheme to analyze one matrix per procedure No negative feedback was provided by the collaborating laboratories in regard to the performance of the candidate method Several collaborating laboratories expressed questions in regard to the AOAC study design of the collaborative study others expressed concern with analyzing 375 g test portions The concern with handling the larger test portions may have contributed to errors observed during testing that resulted in data not used in the statistical analysis During testing four different laboratories detected the presence of Salmonella spp in seven raw ground beef uninoculated control test portions Additionally four different laboratories detected the presence of Salmonella spp in 15 wet pet food uninoculated control test portions Due to detecting positive samples in the control test portions the data provided by these laboratories were not included during the statistical analysis A root cause investigation to determine the source of contamination yielded the following possibilities Due to the high number of samples analyzed including test portions inoculated at a
29. he 3M MDA Salmonella method with all test portions confirming negative For test portions analyzed by the FDA BAM method 132 out of 132 high inoculum and 70 out of 132 low inoculum test portions confirmed positive For the uninoculated controls 0 out of 132 test portions confirmed positive For the low level inoculum a dLPOD value of 0 04 with 95 confidence intervals of 0 16 0 09 was obtained between the 3M MDA Salmonella method and the FDA BAM method The confidence intervals obtained for dLPOD indicated no significant difference between the two methods A dLPOD p value of 0 00 with 95 confidence intervals of 0 13 0 13 was obtained between presumptive and confirmed 3M MDA Salmonella results The confidence intervals obtained for dLPOD p indicated no significant difference between the presumptive and confirmed results using either confirmation process For the high level inoculum a dLPOD value of 0 01 with 95 confidence intervals of 0 04 0 02 was obtained between the 3M MDA Salmonella method and the FDA BAM method The confidence intervals obtained for dLPOD indicated no significant difference between the two methods A dLPODc p value of 0 00 with 95 confidence intervals of 0 03 0 03 was obtained between presumptive and confirmed 3M MDA Salmonella results The confidence intervals obtained for dLPOD p indicated no significant difference between the presumptive and confirmed results Detailed r
30. in Tables 2 and 3 Tables 2013 09A and B summarize the interlaboratory results for all foods tested including POD statistical analysis 10 The results of the collaborating laboratories APC analysis for each matrix are presented in Table C of the Appendix Raw Ground Beef 25 g Test Portions Raw ground beef test portions were inoculated at a low and high level and were analyzed Table 2 for the detection of JOURNAL OF AOAC INTERNATIONAL VOL 96 NO 6 2013 7 Table 1 Participation of each collaborating laboratory Raw ground beef Wet dog food Lab 25 g test portions 375 g test portions 1 Y Y 2 Y Y 3 N Y 4 N Y 5 N Ye 6 N 7 N 8 N 9 Y 10 Y Ys 11 Y Y 12 ye ys 13 Y Y 14 Y Y 15 Y Y 16 Y Y 17 Y N 18 ye N 19 ye N 20 Y N 2 Y Collaborator analyzed the food type N collaborator did not analyze the food type Data obtained from additional shipment of raw ground beef Initial shipment of raw ground beef was not used for evaluation purposes and therefore the data has not been presented Results were not used in statistical analysis due to laboratory error or uninoculated control test portions were confirmed as Salmonella Salmonella spp Uninoculated controls were included in each analysis The results presented for the raw ground beef were from a second shipment of test portions to the collaborating laboratories The initial shipment of raw ground beef test portions sent to collaborators
31. ize Enrichment broth Enrichment Sample matrix g volume mL time h Raw ground beef 27 fat 25 225 18 24 Raw shrimp 25 225 18 24 Bagged spinach 25 225 18 24 Pasteurized liquid whole 100 900 18 24 egg Cooked breaded chicken 325 2925 18 24 Wet pet food dog beef 375 3375 18 24 cuts in gravy canned 6 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 96 No 6 2013 Figure 2013 09A Figure 2013 09B Sample Lysis to mix Suspension has to flow freely inside the tube See Figure 2013 09A Verify that the temperature of the 3M Molecular Detection Heat Block Insert is at 100 1 C Place the rack of LS tubes in the 3M Molecular Detection Heat Block Insert and heat for 15 1 min An alternative to using dry heat for the lysis step is to use a water bath at 100 1 C Ensure that sufficient water is used to cover up to the liquid level in the LS tubes Place the rack of LS tubes in the water bath at 100 1 C and heat for 15 1 min Samples that have not been properly heat treated during the assay lysis step may be considered a potential biohazard and should not be inserted into the 3M Molecular Detection Instrument Remove the rack of LS tubes from the heating block and allow to cool in the 3M Molecular Detection Chill Block Insert for 10 1 min Remove the rack lid during incubation on the 3M Molecular Detection Chill Block Insert The LS solution may freeze when processing less than 48 LS tubes Freezing of the LS solu
32. lla Method First Action 2013 Applicable to detection of Salmonella in raw ground beef 25 g processed breaded chicken 325 g liquid egg 100 g shrimp 25 g fresh spinach 25 g and wet dog food 375 g See Tables 2013 09A and B for a summary of results of the inter laboratory study See Appendix Tables A and B for detailed results of the inter laboratory study A Principle The 3M Molecular Detection Assay MDA Salmonella method is intended for use with the 3M Molecular Detection System for the rapid and specific detection of Salmonella spp in food feed and environmental samples after enrichment After enrichment in prewarmed 3M Buffered Peptone Water ISO 3M BPW ISO medium the 3M MDA Salmonella test utilizes loop mediated isothermal amplification to rapidly amplify Salmonella target DNA with high specificity and sensitivity combined with bioluminescence to detect the amplification Presumptive positive results are reported in real time negative results are displayed after the assay is completed JOURNAL OF AOAC INTERNATIONAL VOL 96 NO 6 2013 3 B Apparatus and Reagents Items b g are available as the 3M MDA Salmonella kit from 3M Food Safety St Paul MN a 3M Molecular Detection System Available from 3M Food Safety b 3M MDA Salmonella reagent tubes 12 strips of eight tubes e Lysis solution LS tubes 12 strips of eight tubes d Extra caps 12 strips of eight caps e Negativ
33. n real time negative results are displayed after completion of the assay Prior to the collaborative study the 3M MDA Salmonella method was certified as a Performance Tested Method PTM following the AOAC guidelines for harmonized PTM studies 5 The aim ofthe PTM study was to demonstrate that the 3M MDA Salmonella method could detect Salmonella in selected foods as claimed by the manufacturer For the 3M MDA Salmonella evaluation six matrices were analyzed raw ground beef 25 g processed breaded chicken 325 g liquid egg 100 g shrimp 25 g fresh spinach 25 g and wet dog food 375 g All other Nor over 100 years Salmonella one of the most frequently 2 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 96 No 6 2013 PTM parameters inclusivity exclusivity ruggedness stability and lot to lot variability tested in the PTM studies satisfied the performance requirements for PTM approval The method was awarded PTM certification number 031208 on March 30 2012 The aim of this collaborative study was to compare the 3M MDA Salmonella method to the U S Department of Agriculture USDA Food Safety and Inspection Service FSIS Microbiology Laboratory Guidebook MLG 4 05 6 for raw ground beef and the U S Food and Drug Administration FDA Bacteriological Analytical Manual BAM Chapter 5 7 method for wet dog food Collaborative Study Study Design For this collaborative study two matrices raw ground beef 80
34. ories Inc Baltimore MD Indaue Mello and Maria Ontiberos Mars Petcare US Kansas City MO Jodene Jurgens and Leslie Thompson Aegis North Sioux City SD David Bosco Food Safety Net Services Fresno CA Amit Morey and Sergio Montez Food Safety Net Services San Antonio TX Kyle Newman Venture Laboratories Inc Lexington KY Mary Bandu and Matt Oltman Chestnut Laboratory Springfield MO Robert Brooks ATC Microbiology LLC North Little Rock AR Christine Gwinn and Scott Moosekian Covance Laboratories Inc Battle Creek MI Joey Marchent Gulf Coast Seafood Laboratory FDA Dauphin Island AL Kathleen T Rajkowski USDA Agricultural Research Services Eastern Regional Research Center Food Safety and Technologies Initiative Glenside PA Shaunti Luce The National Food Lab Livermore CA Hondo Dammann and Dorn Clark Jr Marshfield Food Safety Marshfield WI Wendy McMahon and Deena Awad Silliker Inc Crete IL Michelle Kelly and Megan Greenwell Q Laboratories Inc Cincinnati OH References 1 Centers for Disease Control and Prevention October 26 2011 lt http www cdc gov nezved divisions dfbmd gt accessed November 6 2012 2 Hammack Thomas 2011 Salmonella species in Bad Bug Book Foodborne Pathogenic Microorganisms and Natural Toxins 2nd Ed U S Food and Drug Administration College Park MD 3 U S Department of Agriculture Food Safety and Inspection Service May 2007 lt http
35. sults Detailed results of the POD statistical analysis are presented in Table 2013 09A and Figures 1A and B of the Appendix Wet Dog Food 375 g Test Portions Wet dog food test portions were inoculated at a low and high level and were analyzed Table 3 for the detection of Salmonella spp Uninoculated controls were included in each analysis Sixteen laboratories participated in the analysis of this matrix and the results of 11 laboratories were included in the statistical analysis Laboratories 4 5 10 and 16 detected the presence of Salmonella spp in either the candidate or reference method control replicates Because of the potential for error results from these laboratories were excluded from the statistical analysis Laboratory 12 did not submit results due to cross contamination of sample enrichments as reported by the analyst The MPN levels obtained for this test portion with 95 confidence intervals were 0 72 CFU test portion 0 57 0 90 for the low level and 5 34 CFU test portion 3 46 8 24 for the high level For the high level 131 out of 132 test portions were reported as presumptive positive by the 3M MDA Salmonella method with all test portions confirming positive For the low level 65 out of 132 test portions were reported as presumptive positive by the 3M MDA Salmonella method with all test portions confirming positive For the uninoculated controls 1 out of 132 samples produced a presumptive positive result by t
36. tion will not affect your test If freezing is observed allow the LS tubes to thaw for 5 min before mixing Remove the rack of LS tubes from the 3M Molecular Detection Chill Block Insert 3M Molecular Detection Chill Block Tray system Replace the lid on the rack of LS tubes and firmly invert three to five times to mix Suspension has to flow freely inside the tube Firmly tap the lysis tubes rack on the laboratory bench three to five times Place the rack on the laboratory bench Let it sit undisturbed for at least 5 min to allow the resin to settle Do not mix or disturb the resin at the bottom of the tube See Figure 2013 09B J Amplification One reagent tube is required for each sample and the NC Reagent tube strips can be cut to desired tube number Select the number of individual reagent tubes or eight tube strips needed Place reagent tubes in an empty rack Avoid disturbing the reagent pellets from the bottom of the tubes Select one RC tube and place in rack To avoid cross contamination decap one reagent tubes strip at a time and use a new pipet tip for each transfer step Transfer lysate to reagent tubes and RC tube as follows 00 05 00 Transfer each sample lysate into individual reagent tubes first followed by the NC Hydrate the RC tube last Warning Care must be taken when pipetting LS as carry over of the resin may interfere with amplification 1 Use the 3M Molecular Detection Cap Decap Tool Reagent
37. to decap the reagent tubes one strip at a time Discard cap 2 Transfer 20 uL of sample lysate from the upper portion of the fluid in the LS tube into corresponding reagent tube Dispense at an angle to avoid disturbing the pellets Mix by gently pipetting up and down five times 3 Repeat until individual sample lysate has been added to a corresponding reagent tube in the strip 4 Cover the reagent tubes with the provided extra cap and use the rounded side of the 3M Molecular Detection Cap Decap Tool Reagent to apply pressure in a back and forth motion ensuring that the cap is tightly applied Repeat steps to 4 as needed for the number of samples to be tested When all sample lysates have been transferred repeat steps 1 to 4 to transfer 20 uL of NC lysate into a reagent tube Transfer 20 uL of NC lysate into a RC tube Dispense at an angle to avoid disturbing the pellets Mix by gently pipetting up and down five times Load capped tubes into a clean and decontaminated 3M Molecular Detection Speed Loader Tray Close and latch the 3M Molecular Detection Speed Loader Tray lid See Figure 2013 09C Review and confirm the configured run in the 3M Molecular Detection Software Click the start button in the software and select instrument for use The selected instrument s lid automatically opens Place the 3M Molecular Detection Speed Loader Tray into the 3M Molecular Detection Instrument and close the lid to start the assay Results ar
38. toring the 3M MDA z Computer Compatible with the Detection Instrument 3M Molecular C General Instructions a Store the 3M MDA Salmonella kit at 2 8 C Do not freeze Keep kit away from light during storage After opening the kit check that the foil pouch is undamaged If the pouch is damaged do not use After opening unused reagent tubes should always be stored in the resealable pouch with the desiccant inside to maintain stability of the lyophilized reagents Store resealed pouches at 2 8 C for no longer than 60 days Do not use 3M MDA Salmonella past the expiration date b The 3M Molecular Detection Instrument is intended for use with samples that have undergone heat treatment during the assay lysis step which is designed to destroy organisms present in the sample Samples that have not been properly heat treated during the assay lysis step may be considered a potential 4 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 96 No 6 2013 Table 2013 09A POD summary of raw ground beef 25 g results for the 3M MDA Salmonella method Inoculation level Uninoculated Low High Candidate presumptive positive total No of samples analyzed Candidate presumptive CP POD 50 sy Candidate confirmed positive total No of samples analyzed Candidate confirmed CC POD Se sL SR Positive reference samples total No of samples analyzed Reference POD sp si SR dLP
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