3M Molecular Detection Assay Listeria AOAC Study
Contents
1. 8 yA 49 VN YN YN WN WN YN WN WN WN WN W W VN WN YN YN WN YN WN WN WN WN W W VN YN YN YN WN YN WN WN WN WN W W q8 VN WN YN WN WN YN WN WN WN WN W W VN WN WN WN WN WN WN WN WN WN W W VN WN WN WN WN WN WN WN WN WN W W qv gt s E L ZL 66 VWO OVOV SL vl pt pt pt pt pt pt pt pt pt pt pt pt gel cl q LL i S OL y 6 gt 8 pt pt pt pt pt pt pt pt pt pt pt pt pt pt pt pt 9 VN YN WN WN WN YN WN WN WN WN W WN VN WN WN WN WN WN WN2. Candidate confirmed POD CC Sr 0 00 0 00 0 03 0 00 0 00 0 16 0 48 0 40 0 57 0 51 0 45 0 52 1 00 0 97 1 00 0 00 0 00 0 16 SL 0 00 0 00 0 16 0 00 0 00 0 15 0 00 0 00 0 16 SR 0 00 0 00 0 23 0 51 0 46 0 52 0 00 0 00 0 23 P value 1 0000 0 8762 1 0000 Positive reference samples total No of samples analyzed 0 132 73 132 132 132 Reference POD Sr 0 00 0 00 0 03 0 00 0 00 0 16 0 55 0 47 0 64 0 50 0 45 0 52 1 00 0 97 1 00 0 00 0 00 0 16 SL 0 00 0 00 0 16 0 00 0 00 0 18 0 00 0 00 0 16 SR 0 00 0 00 0 23 0 50 0 45 0 52 0 00 0 00 0 23 P value 1 0000 0 6678 1 0000 dLPOD candidate vs reference 0 00 0 03 0 03 0 07 0 19 0 06 0 00 0 03 0 03 dLPOD candidate presumptive vs candidate confirmed 0 01 0 02 0 04 0 01 0 12 0 13 0 00 0 03 0 03 Results include 95 confidence intervals s Repeatability SD s_ Among laboratory SD Sr Reproducibility SD P value Homogeneity test of laboratory PODs B Apparatus and Reagents Items a and h 0 are available from 3M Food Safety St Paul MN Items b g are available as the 3M MDA Listeria kit from 3M Food Safety a 3M Molecular Detection Instrument b 3M MDA Listeria reagent tubes Twelve strips of eight tubes c Lysis solution LS tubes Twelve strips of eight tubes d Extra caps Twelve strips of eight caps e Ne
3. LPOD the reference method LPODz and the difference in the confirmed candidate and reference methods dLPOD A dLPOD confidence interval not containing the point zero would indicate a statistically significant difference between the 3M MDA Listeria and the AOAC 993 12 reference methods at the 5 probability level In addition to POD the repeatability SD s the among laboratory repeatability SD s1 the reproducibility standard deviation sp and the Py value were calculated The s provides the variance of data within one laboratory the s provides the difference in SD between laboratories and the sp provides the variance in data between different laboratories The Py value provides information on the homogeneity test of laboratory PODs 11 AOAC Official Method 2014 06 Listeria species in Selected Foods and Environmental Surfaces 3M Molecular Detection Assay MDA Listeria Method First Action 2014 Applicable to detection of Listeria species in selected foods including beef hot dogs 25 g deli turkey 25 g cold smoked salmon 25 g full fat cottage cheese 25 g and two environmental surfaces sealed concrete sponge in 100 mL and sponge in 225 mL enrichment volume and stainless steel and sponge in 225 mL enrichment volume enriched in prewarmed DF broth base See Table 2014 06A for a summary of results of the interlaboratory study supporting acceptance of the method See Appendix available on the J AOAC Int w
4. Amanda Thielen Tyson WBA Analytical Springdale AR Leslie Thompson VANGUARD SCIENCES North Sioux City SD Chris Lopez Alex Brandt and Bharath Brahmanda Food Safety Net Services San Antonio TX Luci Hardrath AgSource Laboratories Marshfield WI Yi Chen and Anna Laasri U S Food and Drug Administration Center for Food Safety and Applied Nutrition College Park MD Robert Brooks ATC Microbiology LLC North Little Rock AR Diane Wood and Andrew Sweet Maple Leaf Consumer Foods Guelph Ontario Canada Jean Schoeni Covance Inc Madison WI Brian Kupski and and Nicole Cuthbert Silliker Food Science Center Crete IL Ben Bastin Q Laboratories Inc Cincinnati OH We extend a special thanks to the following team members at Q Laboratories Inc for their efforts during the collaborative study M Joseph Benzinger Jr Allison Mastalerz Kiel Fisher Kateland Koch Will Judd and Nicole Klass References 1 Chen Y 2012 Listeria monocytogenes in Bad Bug Book Foodborne Pathogenic Microorganisms and Natural Toxins 2nd Ed U S Food and Drug Administration Silver Spring MD 2 Centers for Disease Control and Prevention http www cdc gov listeria accessed September 2014 3 New Zealand Ministry of Health May 2001 Listeria monocytogenes Fact Sheet http www foodsafety govt nz elibrary industry Listeria Monocytogenes Science_Research pdf accessed December 2014 4 Heisick J E Rosas Marty L I a
5. 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 No 081203 on March 30 2012 A method modification and matrix extension study was performed in 2014 with the following matrixes beef hot dogs 25 g deli turkey 25 g cold smoked salmon 25 g full fat cottage cheese 25 g bagged raw spinach 25 g whole cantaloupe whole melon sealed concrete sponge in 100 mL and sponge in 225 mL enrichment volume and stainless steel sponge in 225 mL enrichment volume using DF broth base without FAC as the primary enrichment and where applicable a secondary enrichment in Fraser broth base without FAC All other 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 modification and matrix extension was awarded PTM approval and license No 081203 on June 30 2014 The purpose of this collaborative study was to compare the reproducibility among different laboratories of the 3M MDA Listeria method to the AOAC Official Method of Analysis OMA 993 12 Listeria monocytogenes in Milk and Dairy Products 6 reference method for full fat 4 milk fat cottage cheese Collaborative Study Study Design In this collaborative study one matrix full fat cottag
6. WN WN WN W W VN WN WN WN WN WN WN WN WN WN W W q8 VN WN YN YN YN YN WN WN WN WN W W VN YN YN YN WN YN WN WN WN WN W W VN WN YN YN WN YN WN WN WN WN W W qv r gt L eu S17 YAN NE cL bb OL 6 8 Z 9 S v r L cl bb OL 6 8 Z 9 S v r L cl bb OL 6 8 Z 9 S v r L qeq suonlod 3sa pazejnoouiunN suonJod 4S3 AB MO7 suoniod 359 ana UBIH 84 yU p asaayo abeyos yeJ 11nJ 10 S NSO1 J03e10qe 09 enpraIpu z a1qel BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 98 NO 4 2015 1001 Each laboratory analyzed 36 test portions for each method 12 inoculated with a high level of Listeria 12 inoculated with a low level of Listeria and 12 uninoculated controls The 3M MDA Listeria method produced 199 presumptive positive results with 196 confirming positive by traditional confirmation There were 205 confirmed positives by the reference method A background screen of the matrix indicated an absence of indigenous Listeria species For each matrix the level of Listeria was determined by MPN determination on the day of initiation of analysis by the coordinating laboratory The individual laboratory and sample results are presented in Table 2 Table A summarizes the interlaboratory results for all foods tested includin
7. confidence interval of 0 19 0 06 was obtained between the 3M MDA Listeria method and the AOAC 993 12 method The confidence interval obtained for dLPOD indicated no significant difference between the two methods A dLPOD p value of 0 01 with a 95 confidence interval of 0 12 0 13 was obtained between presumptive and confirmed 3M MDA Listeria 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 dLPODc value of 0 00 with a 95 confidence interval of 0 03 0 03 was obtained between the 3M MDA Listeria method and the AOAC 993 12 method The confidence interval obtained for dLPOD indicated no significant difference between the two methods A dLPODcp value of 0 00 with a 95 confidence interval of 0 03 0 03 was obtained between presumptive and confirmed 3M MDA Listeria results The confidence interval obtained for dLPODcp indicated no significant difference between the presumptive and confirmed results Detailed results of the POD statistical analysis are presented in Table A and Figures 1A and B of the Appendix Discussion No negative feedback was provided by the collaborating laboratories in regard to the performance of the 3M MDA Listeria Several laboratories reported difficulty in isolating and identifying Listeria colonies on OXA from samples enriched in the DF broth base with
8. identified whether a test portion was positive or negative more than 99 of the time false positive rate of 1 For full fat cottage cheese the collaborative study indicated no statistically significant difference between the candidate method and the reference method or the presumptive and confirmed results of 1002 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 98 No 4 2015 the candidate method were obtained when the POD statistical model was used Recommendations It is recommended that the 3M Molecular Detection Assay Listeria method be adopted as Official First Action status for the detection of Listeria species in selected foods including beef hot dogs 25 g deli turkey 25 g cold smoked salmon 25 g full fat cottage cheese 25 g and two environmental surfaces sealed concrete sponge in 225 mL enrichment volume and stainless steel and sponge in 225 mL enrichment volume enriched in DF broth base without FAC Acknowledgments We extend a sincere thank you to the following collaborators for their dedicated participation in this study Peyman Fatemi and Sharon Spencer Aemtek Inc Fremont CA Joel Blumfield EDL Labs Inc Purvis MS Adam Hankins McCoy amp McCoy Laboratories Inc Madisonville KY Rob Dermer Natalie Shipley and James Williams Microbac Laboratories Inc Fayetteville NC Ashley Morris Robert Brooks and Karen Powers Microbac Laboratories Inc Maryville TN Jerry Lynn Picket and
9. tube Preparation of the 3M Molecular Detection Instrument a Launch the 3M Molecular Detection Software and log in b Turn on the 3M Molecular Detection Instrument c 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 temperature of 60 C before inserting the 3M Molecular Detection Speed Loader Tray with reaction tubes 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 J Lysis a Allow the LS tubes to warm up to room temperature 20 25 C 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 1 C incubator for 1 h or at room temperature overnight 16 18 h b Remove the enrichment broth from the incubator and gently agitate the contents c One LS tube is required for each sample and the NC sample 1 LS tube strips can be cut to desired LS tube number Select the number of individual LS tubes or eight tube strips needed Place the LS tubes in an empty rack 2 To avoid cross contamination decap one LS tube strip at a time and use a new pipet tip for each transfer step d Transfer enriched sample to LS tubes as described below Note Transfer
10. BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 98 NO 4 2015 993 MICROBIOLOGICAL METHODS Evaluation of 3M Molecular Detection Assay MDA Listeria for the Detection of Listeria species in Selected Foods and Environmental Surfaces Collaborative Study First Action 2014 06 PATRICK BIRD JONATHAN FLANNERY ERIN CROWLEY JAMES AGIN and DAVID GOINS Q Laboratories Inc 1400 Harrison Ave Cincinnati OH 45214 Lisa MONTEROSO and DEANN BENESH 3M Food Safety Department 3M Center Bldg 260 6B 01 St Paul MN 55144 Collaborators B Bastin J Blumfield B Brahmanda A Brandt R Brooks R Brooks Y Chen N Cuthbert R Dermer P Fatemi A Hankins L Hardrath B Kupski A Laasri C Lopez A Morris J Picket K Powers J Schoeni N Shipley S Spencer A Sweet A Thielen L Thompson J Williams D Wood The 3M Molecular Detection Assay MDA Listeria is used with the 3M Molecular Detection System for the detection of Listeria species in food food related and environmental samples after enrichment The assay utilizes loop mediated isothermal amplification to rapidly amplify Listeria target DNA with high specificity and sensitivity combined with bioluminescence to detect the amplification The 3M MDA Listeria method was evaluated using an unpaired study design in a multilaboratory collaborative study and compared to the AOAC Official Method of Analysis OMA 993 12 Listeria monocytogenes in Milk an
11. C 993 12 reference method 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 Transportations Association Upon receipt samples were held by the collaborating laboratory at refrigeration temperature 3 5 C until the following Monday when analysis was initiated a total of 96 h after inoculation All samples were packed with cold packs to target a temperature of lt 7 C during shipment In addition to each of the test portions and the total plate count sample collaborators also received a test portion for each matrix labeled as temperature control Participants were instructed to obtain the temperature of this portion upon receipt of the package document results on the Sample Receipt Confirmation form provided and fax to the study director The shipment and hold times through 120 h of the inoculated test material had been verified as a QC measure prior to study initiation Test Portion Analysis Each collaborator received 72 test portions of full fat cottage cheese 12 high inoculum 12 low inoculum and 12 uninoculated controls for each method Collaborators followed the appropriate preparation and analysis protocol according to the method specified for the matrix Table 1 B
12. IRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 98 NO 4 2015 995 Table 1 Participation of each collaborating laboratory Lab Full fat cottage cheese 1 Y 2 Y 3 Y 4 y 5 Ye 6 Y T Y 8 Y 9 Y 10 Y 11 Y 12 Y 13 y 14 15 Y a Y Collaborator analyzed the food type gt Results were not submitted to the coordinating laboratory Results were not used in statistical analysis due to laboratory error For the analysis of the test portions by the 3M MDA Listeria method a 25 g portion was enriched with 225 mL of prewarmed 37 1 C DF broth base without FAC homogenized for 2 0 5 min and incubated for 26 2 h at 37 1 C Following enrichment samples were assayed by the 3M MDA Listeria method and confirmed following the standard reference method by streaking an aliquot of the primary enrichment onto Oxford Agar OXA Presumptive positive samples were streaked for isolation on Trypticase Soy Agar with yeast extract TSA ye verified morphologically by Gram stain and biochemically confirmed by hemolysis testing and by VITEK 2 GP Biochemical Identification method AOAC OMA 2012 02 9 or API Listeria Identification System biochemical test kits bioM rieux Lyon France Laboratories utilizing API Listeria kits were also required to conduct catalase and oxidase tests For samples analyzed using the AOAC 993 12 reference method 25 g test portions were enriched in prewarmed 45 2 C selective enric
13. L JOURNAL OF AOAC INTERNATIONAL VOL 98 NO 4 2015 997 C General Instructions a Store the 3M MDA Listeria 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 1 month Do not use 3M MDA Listeria 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 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 Safety Precautions 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 L monocytogenes is of particular concern for pregnant women the aged and the infirmed It is recommended that these groups of concern avoid handling this organism After use the enrichment medium and the 3M MDA Listeria tubes can
14. d Dairy Products reference method for the detection of Listeria species in full fat 4 milk fat cottage cheese 25 g test portions A total of 15 laboratories located in the continental United States and Canada participated Each matrix had three inoculation levels an uninoculated control level 0 CFU test portion and two levels artificially contaminated with Listeria monocytogenes a low inoculum level 0 2 2 CFU test portion and a high inoculum level 2 5 CFU test portion using nonheat stressed cells In total 792 unpaired replicate portions were analyzed Statistical analysis was conducted according to the probability of detection POD model Results obtained for the low inoculum level test portions produced a difference in cross laboratory POD value of 0 07 with a 95 confidence interval of 0 19 0 06 No statistically significant Received January 29 2015 The method was approved by the Expert Review Panel for Microbiology for Food and Environmental Surfaces The Expert Review Panel for Microbiology for Food and Environmental Surfaces invites method users to provide feedback on the First Action methods Feedback from method users will help verify that the methods are fit for purpose and are critical to gaining global recognition and acceptance of the methods Comments can be sent directly to the corresponding author or methodfeedback aoac org Supplemental Tables and Figures are available on the J AOAC Int website htt
15. d answer any questions from the participating laboratories Preparation of Inocula and Test Portions The L monocytogenes culture used in this evaluation was propagated in 10 mL of Brain Heart Infusion broth from a frozen stock culture stored at 70 C at Q Laboratories Inc The broth was incubated for 18 0 5 h at 35 1 C Appropriate dilutions of the culture were prepared based on previously established growth curves for both the low and high inoculation levels The full fat cottage cheese was inoculated at a low and high inoculation level with the diluted inoculum and thoroughly hand mixed to ensure an even distribution of microorganisms The inoculated test product was divided into separate 30 g portions which were packaged into sterile Whirl pak bags To determine the level of L monocytogenes in the full fat cottage cheese a five tube most probable number MPN was conducted on the day of initiation of analysis From both the high and low inoculated batches 5 x 50 g test portions the reference method test portions from the collaborating laboratories and 5 x 10 g test portions were analyzed Each test portion was enriched at a 1 10 dilution and evaluated following the AOAC 993 12 reference method The MPN and 95 confidence intervals were calculated from the high medium and low levels using the LCF MPN Calculator Version 1 6 provided by AOAC Research Institute 8 Confirmation of the samples was conducted according to the AOA
16. e cheese was analyzed using 25 g test portions The full fat cottage cheese was obtained from a local retailer and screened for the absence of Listeria by the AOAC 993 12 reference method prior to analysis The matrix was artificially contaminated with nonheat stressed cells of L monocytogenes American Type Culture Collection ATCC Manassas VA 19114 at two inoculation levels 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 were also included at 0 CFU test portion Twelve replicate portions from each of the three inoculation levels were analyzed Two sets of samples 72 total were sent to each laboratory for analysis by 3M MDA Listeria and AOAC 993 12 due to the different sample enrichment procedures for each method Additionally collaborators were sent a 30 g test portion and instructed to conduct a total aerobic plate count using 3M Petrifilm Aerobic Count Plate AOAC OMA 990 12 7 on the day samples were received for the purpose of determining the total aerobic microbial load A detailed collaborative study packet outlining all necessary information related to the study including media preparation test portion preparation and documentation of results was sent to each collaborating laboratory prior to the initiation of the study A conference call was conducted to discuss the collaborative study packet an
17. each enriched sample into individual LS tubes first Transfer the NC last 1 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 2 Transfer 20 uL of sample into an LS tube 3 Repeat step d 2 until each individual sample has been added to a corresponding LS tube in the strip 4 Use the 3M Molecular Detection Cap Decap Tool Lysis to recap the LS tube strip Use the rounded side of the tool to Figure 2014 06B Sample lysis apply pressure in a back and forth motion ensuring that the cap is tightly applied See Figure 2014 06A 5 Repeat steps d d 4 as needed for the number of samples to be tested 6 When all samples have been transferred then transfer 20 uL NC into an LS tube Use the 3M Molecular Detection Cap Decap Tool Lysis tool to recap the LS tube 7 Cover the rack of LS tubes with the rack lid and firmly invert three to five times to mix Suspension has to flow freely inside the tube e 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 wa
18. ebsite for supplementary materials for detailed results of the study com content aoac jaoac interlaboratory http aoac publisher ingentaconnect A Principle The 3M MDA Listeria is intended for use with the 3M Molecular Detection System for the rapid and specific detection of Listeria spp in selected foods and environmental surfaces The 3M MDA uses loop mediated isothermal amplification to rapidly amplify nucleic acid sequences with high specificity and sensitivity combined with bioluminescence to detect the amplification Presumptive positive results are reported in real time while negative results are displayed after the assay is completed Samples are enriched in prewarmed DF broth base which does not contain FAC 996 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 98 No 4 2015 Table 2014 06A Summary of results for the detection of Listeria in full fat cottage cheese 25 g Method 3M MDA Listeria Inoculation level Uninoculated Low High Candidate presumptive positive total No of samples analyzed 1 132 67 132 132 132 Candidate presumptive POD CP so 0 01 0 00 0 04 0 09 0 08 0 16 s 0 00 0 00 0 04 SR 0 09 0 08 0 10 P value 0 4338 Candidate confirmed positive total No of samples analyzed 0 132 0 51 0 42 0 60 0 51 0 45 0 52 0 00 0 00 0 17 0 51 0 46 0 52 0 8931 64 132 1 00 0 97 1 00 0 00 0 00 0 16 0 00 0 00 0 16 0 00 0 00 0 23 1 0000 132 132
19. esults and Interpretation Analgorithm 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 while Negative and Inspect results will be displayed after the run is completed Presumptive positive results should be confirmed using one s preferred method or as specified by the U S Food and Drug Administration Bacteriological Analytical Manual U S Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook AOAC Official Method 993 12 or ISO 11290 methods starting from the 3M primary enrichment followed by transfer to a secondary enrichment or direct plating onto media through 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 Molecular Assay Listeria 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 one s preferred method or as specified by local regulations Results of C
20. g POD statistical analysis 11 As per criteria outlined in Appendix J of the AOAC validation guidelines fractional positive results were obtained Detailed results for each laboratory are presented in Table A and Figures 1A and 1B of the Supplementary Materials The results for each collaborating laboratory s 3M Petrifilm Aerobic Count Plate AOAC 990 12 for full fat cottage cheese are presented in Table B of the Supplementary Materials Full Fat Cottage Cheese 25 g Test Portions Full fat cottage cheese test portions were inoculated at a low and high level and were analyzed for the detection of Listeria spp Table 2 Uninoculated controls were included in each analysis Laboratories 4 and 5 did not submit results to the coordinating laboratory Laboratories 6 and 13 reported deviations in the protocol Laboratory 6 incorrectly incubated their MDA test portions at 30 C for 48 h instead of the required 37 C for 24 h Laboratory 13 confirmed all colony growth regardless of supplementary tests Gram stain catalase reaction indicating that the organism would not be classified as Listeria Gram negative or Gram positive with spores catalase negative and results from these laboratories were excluded from the statistical analysis The MPN levels obtained for the inoculated samples with 95 confidence intervals were 0 80 CFU test portion 0 63 1 00 for the low level and 4 83 CFU test portion 3 30 7 70 for the high level For the hig
21. gative control NC One vial 2 mL f Reagent control RC Two pouches Each pouch contains eight reagent tubes g Quick Start Guide h 3M Molecular Detection Speed Loader Tray i 3M Molecular Detection Chill Block Tray and Chill Block Insert j 3M Molecular Detection Heat Block Insert k 3M Molecular Detection Cap Decap Tool Reagent for reagent tubes 1 3M Molecular Detection Cap Decap Tool Lysis for lysis tubes A confidence interval for dLPOD that does not contain the value 0 indicates a statistical significant difference between the two methods m Empty lysis tube rack n Empty reagent tube rack 0 DF broth base Formulation equivalent to ISO 11290 1 1996 p Disposable pipet Capable of 20 uL q Multichannel 8 channel pipet Capable of 20 uL r Sterile filter tip pipet tips Capable of 20 uL s Filter Stomacher bags Seward Ltd West Sussex UK or equivalent t Stomacher Seward 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 storing the 3M MDA z Computer Compatible with the 3M Molecular Detection Instrument BIRD ET A
22. h level 132 out of 132 test portions LPOD p of 1 00 were reported as presumptive positive by the 3M MDA Listeria method with all 132 test portions LPOD c of 1 00 confirming positive Based on the valid data submitted from each of the collaborating laboratories 0 false negative results or false positive results were obtained resulting in 132 confirmed positives LPOD of 1 00 For the low level 67 out of 132 test portions LPODcp of 0 51 were reported as presumptive positive by the 3M MDA Listeria method with 64 test portions LPOC c of 0 48 confirming positive Based on the valid data submitted from each of the collaborating laboratories three false positive results were obtained resulting in 64 confirmed positives LPOD of 0 48 For the uninoculated controls one out of 132 samples LPOD p of 0 01 produced a presumptive positive result by the 3M MDA Listeria method with all 132 test portions LPOD of 0 00 confirming negative Based on the valid data submitted from each of the collaborating laboratories 0 false negative results and 1 false positive results were obtained resulting in 0 confirmed positives LPOD of 0 00 For test portions analyzed by the AOAC 993 12 Method 132 out of 132 high inoculum test portions and 73 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 07 with a 95
23. hment broth homogenized for 2 0 5 min and incubated at 30 2 C for 48 2 h Samples were streaked onto OXA and presumptive positive samples were streaked for isolation onto TSA ye Colonies from TSA ye were verified morphologically by Gram stain and biochemically confirmed by hemolysis test and by VITEK 2 GP Biochemical Identification method or API Listeria biochemical test kits Laboratories utilizing API Listeria kits were also required to conduct catalase and oxidase tests Statistical Analysis Each collaborating laboratory recorded results for the reference method and the 3M MDA Listeria method on the data sheets provided The data sheets were submitted to the study director at the end of testing for analysis The results of each test portion for each sample were compiled by the study director and the 3M MDA Listeria results were compared to the reference method for statistical analysis Data for each test portion size were analyzed using the probability of detection POD 10 The POD was calculated as the number of positive outcomes divided by the total number of trials The cross laboratory POD LPOD was calculated for the candidate presumptive results LPODcp the candidate confirmatory results including false negative results LPODcc the difference in the candidate presumptive and confirmatory results dLPOD p presumptive candidate results that confirmed positive excluding false negative results
24. isothermal amplification of target nucleic acid sequences to detect Listeria in enriched food feed and environmental samples The isothermal amplification is a PCR conducted at a constant temperature eliminating the need for temperature cycling and decreasing the time to results The 3M MDA Listeria method allows for the rapid and specific detection of Listeria species after as little as 24 h of enrichment using prewarmed 37 1 C Demi Fraser DF broth base without ferric ammonium citrate FAC or 3M Modified Listeria Recovery Broth mLRB After enrichment samples are evaluated using the 3M MDA Listeria on the 3M Molecular Detection System Presumptive positive results are reported in real time while negative results are displayed after completion of the assay 75 min isteria is a Gram positive rod shaped bacterium found 994 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 98 No 4 2015 Prior to the collaborative study the 3M MDA Listeria method was validated according to AOAC guidelines 5 in a harmonized AOAC Performance Tested Method PTM study The objective of the PTM study was to demonstrate that the 3M MDA Listeria method could detect Listeria on selected environmental surfaces as claimed by the manufacturer For the 3M MDA Listeria PTM evaluation three matrixes were evaluated stainless steel sponge in 225 mL 3M mLRB sealed concrete sponge in 225 mL 3M mLRB and plastic swab in 10 mL 3M mLRB All other
25. mp Tatini S R 1995 J Food Prot 58 733 736 5 Official Methods of Analysis 2012 19th Ed Appendix J Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces AOAC INTERNATIONAL Gaithersburg MD http www eoma aoac org app_j pdf accessed July 2014 6 Official Methods of Analysis 1999 Listeria monocytogenes in Milk and Dairy Products AOAC INTERNATIONAL Gaithersburg MD Method 993 12 accessed July 2014 7 Official Methods of Analysis 2002 Aerobic Plate Count in Foods AOAC INTERNATIONAL Gaithersburg MD Method 990 12 accessed July 2014 8 Least Cost Formulations Ltd MPN Calculator Version 1 6 www lcfitd com customer LCFMPNCalculator exe accessed September 2014 9 Official Methods of Analysis 2012 Gram Positive Bacteria Identification AOAC INTERNATIONAL Gaithersburg MD Method 2012 02 accessed July 2014 10 Wehling P LaBudde R Brunelle S amp Nelson M 2011 J AOAC Int 94 335 347 11 Least Cost Formulations Ltd 2011 AOAC Binary Data Interlaboratory Study Workbook http lcfitd com aoac aoac binary v2 3 xls accessed September 2014
26. n Chill Block Insert from the freezer for use remove it and the 3M Molecular Detection Chill Block Tray together Use the 3M Molecular Detection Chill Block Insert 3M Molecular Detection Chill Block Tray within 20 min H Preparation of the 3M Molecular Detection Heat Block Insert 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 2014 06B Enrichment protocols using Demi Fraser broth base without FAC Primary enrichment Demi Fraser broth no FAC Sample matrix Sample size Enrichment broth volume mL Enrichment temperature 1 C Enrichment time h Food Full fat cottage cheese 259 225 37 24 28 Beef hot dogs 259 225 37 24 28 Deli turkey 259 225 37 24 28 Cold smoked salmon 259 225 37 24 28 Environmental surfaces Stainless steel 1 Swab 10 37 26 30 Sealed concrete 1 Sponge 100 37 26 30 Stainless steel sealed concrete 1 Sponge 225 37 26 30 998 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 98 No 4 2015 Figure 2014 06A Transfer of enriched sample to lysis solution
27. ollaborative Study For this collaborative study the 3M MDA Listeria method was compared to the AOAC 993 12 reference method for full fat cottage cheese A total of 15 laboratories throughout the United States and Canada participated in this study with 13 laboratories submitting data for the full fat cottage cheese 1000 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 98 No 4 2015 ynsal aayeBau asjej e Buljeoipul aaijisod pawuyuod yng 81 98 17 YAWN WE uo aayebau ayduns sd sem ajdues 7 nsa aanisod asyjey e Buyjesipul aayeBau pauuos jng evaysi7 YAN WE UO asaysod aAndwnseid sem ajdues 10149 Asoyesoqe 0 anp sis jeue jeoysie s ul pasn jou aJam synsey 2 q paara9a JOU 819M SJNS 1 JO X zew Siy ul ayedionued jou pip 10 e 0q8 YN e dwes ul pajoajap jou ajam salgads eyajs 7 sajduwes ul pajoajep J M sargads EU JSIT GL oe vl Se e E gEl S cl S LL S OL y 6
28. out FAC when compared to samples enriched in the AOAC 991 12 selective enrichment broth This may be related to differences in formulation between the two enrichments The AOAC 993 12 enrichment broth is designed to reduce the background flora on OXA and is more selective than DF broth base without FAC In some instances this level of selectivity may cause stress on Listeria cells thus requiring a longer enrichment time to reach a detectable level Based on the data submitted two laboratories Laboratories 6 and 13 were removed from statistical consideration for the full fat cottage cheese During analysis Laboratory 6 did not follow the approved incubation time and temperature for the candidate method samples were incubated for 48 h at 30 C and the validated enrichment time and temperature are 24 28 h at 37 C and Laboratory 13 confirmed growth from all plates regardless of supplementary tests that would have precluded confirmation via API Listeria test kits bioM rieux Due to this fact all samples confirmed via API Listeria produced a Listeria species result even if Gram stain reaction Gram negative motility reaction negative catalase reaction negative and oxidase reaction positive would indicate the organism is not of the genus Listeria During the analysis of the full fat cottage cheese four false positive results were obtained out of 396 test portions analyzed with the candidate method The 3M MDA Listeria correctly
29. p aoac publisher ingentaconnect com content aoac jaoac Corresponding author s e mail Imonteroso gmmm com DOI 10 5740 jaoacint 15 026 differences were observed in the number of positive samples detected by the 3M MDA Listeria method versus the AOAC OMA method widespread in the environment and one species Listeria monocytogenes is known to be the causative agent of listeriosis in humans 1 Due to its high mortality rate specifically in susceptible individuals such as older adults pregnant women newborns and adults with weakened immune systems listeriosis presents itself as an important health problem in the United States Canada and throughout the world 2 Listeria s ability to survive in extreme conditions such as low temperature and a broad pH range 4 4 to 9 4 can cause severe problems for food manufacturers as the organism can survive cleaning conditions and contaminate food commodities 1 3 While less frequent than other foodborne pathogens outbreaks from L monocytogenes have been linked to a wide variety of food types such as raw milks and cheeses pasteurized dairy products smoked seafood ready to eat deli meats hot dogs and most recently cantaloupes 2 The presence of other Listeria species such as L innocua L welshimeri or L ivanovii is often used as an indicator for the possible contamination of L monocytogenes 4 The 3M Molecular Detection Assay MDA Listeria method uses loop mediated
30. potentially contain pathogenic materials When testing is complete follow current industry standards for the disposal of contaminated waste Consult the Material Safety Data Sheet MSDS for additional information and local regulations for disposal Ethanol used in the method is flammable and caution should be used Consult MSDS for additional information E Sample Enrichment a Prewarm DF broth base without FAC to 37 1 C b Aseptically combine the enrichment medium and sample following the procedures in Table 2014 06B For all meat and highly particulate samples the use of filter bags is recommended Homogenize thoroughly Stomacher blender for 2 0 5 min Incubate at 37 1 C F Preparation of the 3M Molecular Detection Speed Loader Tray a 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 b Rinse the 3M Molecular Detection Speed Loader Tray with water c Use a disposable towel to wipe the 3M Molecular Detection Speed Loader Tray dry d Ensure that the 3M Molecular Detection Speed Loader Tray is dry before use G Preparation of the 3M Molecular Detection Chill Block Insert Before using the 3M Molecular Detection Chill Block Insert ensure that 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 Detectio
31. sired tube number Select the number of individual reagent tubes or eight tube strips needed 2 Place reagent tubes in an empty rack 3 Avoid disturbing the reagent pellets from the bottom of the tubes b Select one RC tube and place in rack c To avoid cross contamination decap one reagent tube strip at a time and use a new pipet tip for each transfer step d Transfer lysate to reagent tubes and RC tube as described below Note 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 carryover of the resin may interfere with amplification 1 Use the 3M Molecular Detection Cap Decap Tool Reagent to decap the reagent tubes one reagent tube 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 step d 2 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 5 Repeat steps d d 4 as needed for the number of samples to be tested 6 When all sample l
32. ter 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 f 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 The LS solution may freeze when processing fewer than 48 LS tubes Freezing of the LS solution will not affect the test If freezing is observed allow the LS tubes to thaw for 5 min before mixing Remove the rack lid during incubation on the 3M Molecular Detection Chill Block Insert g 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 h Firmly tap the lysis tubes rack on the laboratory bench three to five times i Place the rack on the laboratory bench and let sit undisturbed for 5 10 min to allow the resin to settle Do not mix or disturb the resin at the bottom of the tube See Figure 2014 06B 00 05 00 BIRD ET AL JOURNAL OF AOAC INTERNATIONAL VOL 98 NO 4 2015 999 Figure 2014 06C Transfer of lysate to reagent tube K Amplification a One reagent tube is required for each sample and the NC 1 Reagent tube strips can be cut to de
33. ysates have been transferred repeat steps d d 4 to transfer 20 uL NC lysate into a reagent tube 7 Transfer 20 uL NC lysate into an RC tube Dispense at an angle to avoid disturbing the pellets Mix by gently pipetting up and down five times e 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 Figure 2014 06C f Review and confirm the configured run in the 3M Molecular Detection Software g Click the Start button in the software and select instrument for use The selected instrument s lid automatically opens h Place the 3M Molecular Detection Speed Loader Tray into the 3M Molecular Detection Instrument and close the lid to start the assay Results are provided within 75 min although positives may be detected sooner i After the assay is complete remove the 3M Molecular Detection Speed Loader Tray from the 3M Molecular Detection 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 and away from the assay preparation area L R
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