CERTIFICATION AOAC Performance Tested SM Certificate No. 100901 The AOAC Research Institute hereby certifies that the performance of the test kit known as: E. coli O157:H7 Assay Kit manufactured by BioFire Defense, LLC 79 West 4500 South, Suite 14 Salt Lake City, Utah 84107 This method has been evaluated in the AOAC Performance Tested Methods SM Program, and found to perform as stated by the manufacturer contingent to the comments contained in the manuscript. This certificate means that an AOAC Certification Mark License Agreement has been executed which authorizes the manufacturer to display the AOAC Performance Tested SM certification mark along with the statement "THIS METHOD'S PERFORMANCE WAS REVIEWED BY AOAC RESEARCH INSTITUTE AND WAS FOUND TO PERFORM TO THE MANUFACTURER'S SPECIFICATIONS" on the above mentioned method for a period of one calendar year from the date of this certificate (January 14, 2016 December 31, 2016). Renewal may be granted at the end of one year under the rules stated in the licensing agreement. Deborah McKenzie Deborah McKenzie, Senior Director Signature for AOAC Research Institute January 14, 2016 Date 2275 Research Blvd., Suite 300, Rockville, MD 20850 3250 USA * Telephone: +1 301 924 7077 * Fax: +1 301 924 7089 Internet e mail: aoacri@aoac.org * World Wide Web Site: http://www.aoac.org
METHOD AUTHORS Jenkyn Powell, Kelly Winterberg, Haleigh Millward, and Stephanie Thatcher SUBMITTING COMPANY Idaho Technology 390 Wakara Way Salt Lake City, UT 84108 USA CURRENT COMPANY BioFire Defense, LLC 79 West 4500 South, Suite 14 Salt Lake City, Utah 84107 USA KIT NAME(S) E. coli O1257:H7 CATALOG NUMBERS ASAY ASY 0118, ASAY ASY 0128 INDEPENDENT LABORATORY Food Safety Net 199 W. Rhapsody San Antonio, TX 78216 USA Marshfield Food Safety Marshfield Clinic 1000 N. Oak Ave. Marshfield, WI 54440 USA AOAC EXPERTS AND PEER REVIEWERS Thomas Hammack 1, Yi Chen 2, Joseph Odmeru 3, Michael Brodsky 4 1, 2 US Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, MD, USA 3 University of Guelph, Guelph, Ontario, Canada 4 Brodsky Consultants, Thornhill, ON, Canada APPLICABILITY OF METHOD Target organism E. coli O157:H7 (genetically) Matrices Original Validation (25 g ) USDA FSIS 5.04 Raw ground beef FDA BAM Ch 4a uncooked spinach Matrix Extension 2010 (375 g) ground beef, beef trim Performance claims The E. coli O157:H7 LT FSS method is applicable for the specific detection of E. coli O157:H7 in ground beef and spinach. The E. coli O157:H7 LT FSS is at least as sensitive as the USDA MLG (3) and FDA BAM (4) methods at low inoculum levels of approximately 1 colony forming unit (CFU) in 25 g of food sample with individual and pooled samples. REFERENCE METHODS United States Department of Agriculture/Food Safety Inspection Services Microbiological Laboratory Guidebook http://www.fsis.usda.gov/pdf/mlg_5_04.pdf (3) United States Food and Drug Administration, FDA Bacteriological Analytical Manua lhttp://www.cfsan.fda.gov/~ebam/bam 4a.htm (4) PRINCIPLE OF THE METHOD The E. coli O157:H7 LT FSS is a qualitative polymerase chain reaction (PCR) detection method for E. coli O157:H7 in food. The result is determined as soon as 9 hours after sampling. Results are reported as positive or negative. This rapid detection method couples real time PCR with a short enrichment time. The sensitivity of PCR allows for a shorter enrichment time than standard detection methods. Real time PCR specifically amplifies target DNA which is detected by fluorescent probes. All required PCR components including target specific fluorescent probes are included in freeze dried reagent vials for ease of use. Enrichment for this system uses commercially available media (Buffered Peptone Water). Approximately one CFU of E. coli O157:H7 in 25 g of food can be detected after just 8 hours of enrichment for raw ground beef or uncooked spinach (9 hours for post enrichment pooling). The R.A.P.I.D. LT instrument has an advantage over traditional PCR instruments because it uses air thermocycling to heat and cool the sample (within a closed reaction vessel) instead of a metal block, allowing for more rapid heating and cooling. The method involves a series of sequential steps: sample collection and enrichment, sample processing which includes cell lysis to release DNA, DNA amplification (PCR) in the Idaho Technology R.A.P.I.D. LT instrument, and automatic result interpretation by the R.A.P.I.D. LT FSS software. Critical components of the E. coli O157:H7 LT FSS system: o Sample enrichment: single 8 9 hour enrichment in liquid media (Buffered Peptone Water). o Sample pooling of up to five samples (optional): Pooling allows the end user to test five samples with one reagent vial. Postenrichment pooling is accomplished by combining 50 ml of each of five enriched samples to create a single 250 ml composite pooled sample. If a pooled sample is positive for E. coli O157:H7, the original enriched samples can be retested individually to determine which sample is positive. o Sample processing: The enriched sample is tested directly by PCR after a quick cell lysis step. A small amount of enriched sample is added to a bead tube (included in the E. coli O157:H7 LT kit) for physical lysis. The ceramic beads in the bead tube physically disrupt the cells to release DNA when shaken vigorously in the Disruptor Genie. PRINCIPLE OF THE METHOD Cont. o E. coli O157:H7 specific freeze dried reagents: E. coli O157:H7 LT reagent vials contain all of the components required for real time PCR on the R.A.P.I.D. LT instrument. Each vial contains a dried pellet that is easily reconstituted with buffer and lysed sample. Specific primers are included that amplify only E. coli O157 targets. As the target sequence is amplified, fluorescent labeled probes recognize only the amplified E. coli O157:H7 target. This provides an additional level of specificity. The fluorescent signal is detected by the R.A.P.I.D. LT instrument. o Internal control: The E. coli O157:H7 LT assay contains an internal amplification control (AC). The AC demonstrates whether the conditions inside the reaction are sufficient for successful PCR. The AC is detected in a different fluorescent channel than the target. The target is detected by a probe that fluoresces at 640 nm (Channel 2 on the instrument), while the AC is detected by a probe that fluoresces at 705 nm (Channel 3 on the instrument). o Rapid real time PCR: The R.A.P.I.D. LT instrument is an air thermocycler that rapidly alternates between the hot and cool temperatures required for PCR. PCR is sensitive because it can theoretically amplify a single copy of target DNA sequence. The E. coli O157:H7 LT FSS is specific due to target specific primers and fluorescent probes. o Melt detection: A post PCR melting procedure increases both sensitivity and specificity of the method. The fluorescent probe used for detection is melted from the PCR product to produce a peak that is used for detection, and shifts with changes in target sequence. Specific detection of E. coli O157:H7 requires a probe to melt within a specific temperature range. Thus, it is distinguished from other strains of E. coli that amplify. o Automated answers: The E. coli O157:H7 LT FSS includes easy to use software that operates the instrument, automatically collects and analyzes data, and reports positive or negative results in an easyto read, printable format. Total run time for the E. coli O157:H7 LT FSS on the R.A.P.I.D. LT is about 1 hour rather than 2 3 hours for other PCR instruments.
BioFire E. coli AOAC Certification Number 100901 DISCUSSION OF THE VALIDATION STUDY Comparison to Reference Methods: The E. coli O157:H7 LT FSS results were equivalent to or better than results for reference methods at low inoculum levels. Of the 60 total samples evaluated with the test kit (for both individual and pooled), 39 samples (65%) were detected as positive with the E. coli O157:H7 LT FSS. Of the 60 samples evaluated with the reference methods, 22 samples (37%) were positive. The average false positive rate was 0%, and average false negative rate was 1%. Raw Ground Beef Results from tests with raw ground beef demonstrated that the E. coli O157:H7 LT FSS was significantly different than or equivalent to the USDA MLG method (1). In the tests performed at the sponsor lab the FSS method detected 10 confirmed positives in both individual and pooled tests while the MLG method only detected 2 positive samples. Independent laboratory results for ground beef were similar for individual tests, 11 positive samples were detected and confirmed by the FSS method whereas only 5 were detected by the reference method. The reference method used by the independent laboratory included a presumptive screen for positive samples using a PCR test. The pooled samples tested by the independent laboratory revealed 1 additional positive sample which was confirmed. The additional positive detected and confirmed may indicate either a very low presence of E. coli O157:H7 in the original sample that was detected after a longer incubation of 9 hours required for pooled samples or possible cross contamination by the independent lab. Uncooked Spinach Results from tests with uncooked spinach demonstrated that the E. coli O157:H7 LT FSS is statistically equivalent to the FDA BAM method (2). The only false negative sample observed in this study was a pooled spinach sample. Even with this false negative, the pooled spinach samples had a X 2 of 0.61. Inclusivity and Exclusivity: The E. coli O157:H7 LT FSS is highly specific for E. coli O157:H7. Out of the 60 strains that were tested for inclusivity (one strain of the original 61 could not be revived from frozen culture, isolate FS 574) all 60 were detected, including two non motile (NM) strains that are genetically H7. It did not detect any of the 45 strains used in the exclusivity panel, including closely related E. coli O157 strains. Ruggedness and Reagent Variation: The E. coli O157:H7 LT FSS is robust and reproducible as demonstrated by the ruggedness, lot to lot and shelf life studies. The ruggedness study demonstrated that the system produced consistent results with user introduced variability in reagent preparation time and sample volumes. The lot to lot and shelf life study demonstrated that the E. coli O157:H7 LT FSS gave consistent results with three lots of reagents produced at different times, including one lot close to the age of expiration. REFERENCES CITED 1. Powell, J, Winterberg, K., Millward, H., and Thatcher, S., Evaluation of the E. coli O157:H7 Assay Kit, AOAC Performance Tested SM certification number 100901. 2. AOAC Research Institute Validation Outline for E. coli O157:H7 Assay Kit, Approved October 2009. 3. United States Department of Agriculture/Food Safety Inspection Services Microbiological Laboratory Guidebook http://www.fsis.usda.gov/pdf/mlg_5_04.pdf 4. United States Food and Drug Administration, FDA Bacteriological Analytical Manua lhttp://www.cfsan.fda.gov/~ebam/bam 4a.htm 5. Johnson, J.R., and Stell, A.L. 2001. PCR for specific detection of H7 flagellar variant of flic among extraintestinal pathogenic Escherichia coli. J. Clin. Microbiol. 39:3712 7. 2
Table I: Method Comparison Results Reference Method Test kit Test Kit Performance Matrix Raw Chicken Cooked Ham Chocolate Inoculating organism Typhimurium Enteritidis Senftenberg Level MPN CFU/25g No. test portions Presump. Confirmed Chi square Sensitivity rate, % False negative Specificity rate, % Low <0.8 20 13 13 13 100 0 100 0 Control 0 5 0 0 0 Low <0.8 20 7 7 7 100 0 100 0 Control 0 5 0 0 0 High B 10.8 20 17 17 17 100 0 100 0 Low C 0.9 20 9 9 9 100 0 100 0 Low A <0.8 20 1 1 1 100 0 100 0 Control 0 15 0 0 0 False positive Table II. Method Comparison Results, Pooled Samples Reference Method Test kit Test Kit Performance Matrix Raw Chicken Cooked Ham Chocolate Inoculating organism Typhimurium Enteritidis Proto col MPN CFU/25g No. test portions Presump. Confirmed Chi square Sensitivity rate, % False negative Specificity rate, % PCR 18.8 10 10 10 10 100 0 100 0 PTX 18.8 10 10 10 10 100 0 100 0 PCR 5.8 10 10 10 10 100 0 100 0 PTX 5.8 10 10 10 10 100 0 100 0 PCR 10.8 10 9 9 9 100 0 100 0 Senftenberg PTX 10.8 10 9 9 9 100 0 100 0 PTX = PATHATRIX with PCR protocol False positive
Table III: Inclusivity Results # Somatic Group Genus Sub Species/ Serovar Source BPW PCR BPW PTX NFDM PCR NFDM PTX 1 Arizonae CDC 346 86 + + + 2 Salamae ATCC 15786 + + + + 3 51 Hilversum ATCC 15784 + + + + 4 51 Treforest CDC 657 + + + + 5 53 Bockenheim CDC 1097 + + + 6 55 Tranora CDC 857 + + + + 7 57 Locarno CDC 886 + + + + 8 59 Betioky CDC 946 + + + 9 66 Malawi CDC 327 80 + + + + 10 66 Maregrosso CDC 235 77 + + + + 11 67 Crossness CDC 1795 + + + + 12 A Paratyphi A CDC 1072 82 + + + 13 A Paratyphi A ATCC 9150 + + + + 14 B Abony NCTC 6017 + + + + 15 B Agona green beans + + + + 16 B Brandenburg Food environmental + + + + 17 B Bredeney M & B Meal + + + + 18 B California ATCC 23201 liver of turkey + + + + 19 B Derby M & B Meal + + + + 20 B Heidelberg ATCC 8326 + + + + 21 B Indiana CDC 343 86 + + + + 22 B Paratyphi B ATCC 8759 + + + + 23 B Saintpaul Whey + + + + 24 B Sandiego ATCC 23199 NG NG NG NG 25 B Schwarzengrund M & B Meal + + + + 26 B Typhimurium Milk + + + + 27 B Typhimurium Ice Cream + + + + 28 B Typhimurium Liquid Egg + + + + 29 B Suc pos Typhimurium ATCC 6994 + + + 30 B Vellore ATCC 15611 + + (BC) + 31 C1 Bareilly pasta + + + + 32 C1 Bornum Ice cream + + + + 33 C1H2Sneg Choleraesuis ATCC 10708 + + + Cholaraesuis + + + + 34 C1 Kunzendorf ATCC 12011 35 C1H2Sneg Djugu Food isolate + + + + 36 C1 Infantis CDC 139 84 + + + + 37 C1 Jerusalem tuna meal + + + 38 C1 Livingstone CDC 110 85 + + + + 39 C1 Mbandaka frozen whole egg + + + + 40 C1 Montevideo Food isolate + + + + 41 C1 Nienstedten Pasta + + + + 42 C1 Ohio CDC 299 86 + + + + 43 C1 Oranienburg CDC 447 86 + + + + 44 C1 Paratyphi C ATCC 13428 + + + + 45 C1 Potsdam ATCC 25957 + + + + 46 C1Lac pos Tennessee Peanut Butter crème + + + + 47 C1Lac pos Tennessee Chocolate candy + + + + 48 C1 Thompson Ice Cream + + + + 49 C2 Albany CDC 151 86 + + +
BioFire E. coli AOAC Certification Number 100901 50 C2 Bovismorbificans CDC 889 83 + + + + 51 C2 Glostrup ATCC BAA 556 + + + + 52 C2 Hadar Ice cream + + + + 53 C2 Hadar ATCC 51956 (additional) + + + + 54 C2 Kentucky Chocolate + + + + 55 C2 Kottbus CDC 292 85 + + + + 56 C2 Muenchen CDC 427 85 + + + + 57 C2Lac pos Newport CDC 520 86 + + + + 58 C2 Tallahasee ATCC 12002 + + + + 59 D1 Berta CDC 212 86 + + + + 60 D1 Eastbourne Food isolate + + + + 61 D1 Enteriditis Ice Cream + + + + 62 D1 Enteriditis Environmental + + + + 63 D1 Enteriditis ATCC 4931 (additional) + + + + 64 D1 Gallinarum ATCC 9184 NG NG NG NG 65 D1 Gallinarum CDC 2950 79 (additional) (low) + + + 66 D1 Javiana Food isolate + + + + 67 D1 Miami CDC 114 84 Dried yeast + + + + 68 D1 Panama CDC 551 86 + + + + 69 D1 Typhi CDC 2049 87 + + + + 70 D2 Ouakam CDC 32 87 + + + + 71 E1 Anatum Poultry Meal + + + + 72 E1 Anatum ATCC 9270 (additional) + + + + 73 E1 Amsterdam M & B Meal + + + + 74 E1 Amsterdam Food isolate + + + + 75 E1 London CDC 382 84 + + + + 76 E1 Weltervreden CDC 110 83 + + + + 77 E2 Binza Food isolate + + + + 78 E2 Drypool Ice cream + + + + 79 E2 Drypool Gelatin + + + + 80 E2 Manila M & B Meal + + + + 81 E2 Newbrunswick Pasta + + + + 82 E2 Newhaw M & B Meal + + + + 83 E3 Houtenae ATCC 29834 + + + + 84 E3 Illinois ATCC 11646 + + + + 85 E4 Krefeld CDC 939 84 + + + + 86 E4 Senftenberg Chocolate + + + + 87 E4 Simsbury ATCC 12004 + + + + 88 F Abaetetuba Grain product + + + + 89 F Heerlen Feces ATCC 15792 + + + + 90 F Rubislaw Pasta + + + + 91 G Ajiobo M & B Meal + + + + 92 G Cubana Spinach + + + + 93 G Cubana Food ingredient + + + + 94 G Havana Food isolate + + + + 95 G Havana M & B Meal + + + + 96 G Poona ATCC 4840 + + + + 97 G Putten ATCC 15787 + + + + 98 G Roodepoort Pet Food + + + + 99 G Worthington CDC 247 85 + + + + 100 H Caracas Dog food + + + + 101 H Ferlac ATCC 43976 + + + + 2
BioFire E. coli AOAC Certification Number 100901 102 H Madelia CDC 288 85 + + + + 103 H Sundsvall CDC 668 84 + + + + 104 I Gaminara ATCC 8324 + + + + 105 I Saphra CDC 479 85 + + + + 106 II Zwickau ATCC 15805 feces + + + + 107 J Michigan CDC 47 85 + + + + 108 K Cerro Cheese powder + + + + 109 K Cerro ATCC 10723 (additional) + + + + 110 L Minnesota CDC 245 86 + + + + 111 N Urbana Poultry Meal + + + + 112 O Adelaide Poultry Meal + + + + 113 O Adelaide M & B Meal + + + + 114 O Alachua dehydrated turkey + + + + 115 P Inverness CDC 133 87 + + + + 116 R Johannesburg CDC 500 86 + + + + 117 T Weslasco CDC 185 87 + + + + 118 U Houtenae ATCC 15783 + + + + 119 X Bere CDC 315 86 + + + + 120 X Phoenix CDC 696 84 + + + + 121 Z Flint CDC 16 86 + + + + 122 Z Wassenaar CDC 121 87 + + + + 123 bongori ATCC 43975 + + + + BPW = Buffered peptone water, NFDM = Nonfat dry milk, PTX = PATHATRIX Protocol, NG = no growth + = detected, = not detected, BC = Bad call in software Table IV. Exclusivity Results Result Item Genus Species Source 1 Acetobacter aceti ATCC 23746 NG NG 2 Citrobacter amalonaticus Gene Trak 3 Citrobacter freundii ATCC 6879 4 Citrobacter braakii ATCC 29063 5 Citrobacter farmerii ATCC 51112 6 Citrobacter youngae ATCC 29935 7 Enterobacter aerogenes ATCC 35028 8 Enterobacter agglomerans ATCC 29917 9 Enterobacter cloacae ATCC 35030 10 Enterobacter cloacae ATCC 7256 11 Enterobacter sakazakii ATCC 51329 12 Enterococcus faecalis ATCC 49452 13 Enterococcus faecalis ATCC 33186 14 Escherichia blattae ATCC 29907 15 Escherichia coli ATCC 11229 16 Escherichia coli O157:H7 ATCC 35150 17 Hafnia alvei ATCC 25927 18 Klebsiella oxytoca Gene Trak 19 Klebsiella pneumoniae ATCC 9591 20 Morganella morganii spp. morganii ATCC 29853 21 Proteus mirabilis Microbiologics 22 Proteus vulgaris ATCC 8427 23 Providencia stuartii Org Tek, clinical 24 Serratia liquefaciens Microbiologics 25 Serratia marcescens ATCC 14041 26 Shigella boydii Microbiologics 27 Shigella flexineri Microbiologics 28 Shigella sonneii U. Chicago Hosp. PCR PTX 3
BioFire E. coli AOAC Certification Number 100901 29 Staphyloccus aureus ATCC 13565 30 Yersinia enterocolitica ATCC 29913 31 Yersinia frederiksenii Envir.Dairy 32 Yersinia pseudotuberculosis Org Tek, clinical PTX = PATHATRIX Protocol, NG = no growth, + = detected, = not detected ORIGINAL CERTIFICATION DATE October 05, 2009 METHOD MODIFICATION RECORD CERTIFICATION RENEWAL RECORD Renewed Annually through December 2016 SUMMARY OF MODIFICATION Under this AOAC Performance Tested SM License Number, 100901 this method is distributed by: Under this AOAC Performance Tested SM License Number, 100901 this method is distributed as: 4