AOAC Method Comparison Study. Deli Turkey 1 CFU/25g & 5 CFU/25g
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1 AOAC Method Comparison Study Deli Turkey 1 CFU/25g & 5 CFU/25g
2 AOAC Guidelines for Matrix Validation - AOAC mandates two-tiered fractional recovery procedure 1 CFU/25g Low level, recovery target between 25%-75% Results <25% or >75% are considered invalid 5 CFU/25g High Level 100% recovery is required for 5 CFU inoculations Must be run in parallel with reference methods Escherichia coli O157 STEC: FDA BAM 4A Salmonella enterica: USDA MLG 4.08 Listeria monocytogenes: USDA MLG 8.09
3 Experimental Design - Inoculation Experimental Design All bacteria inoculated into matrix and stored at 4 C for 48 hours As per AOAC guidelines Replicates real-world conditions by inducing stress All 3 target organisms inoculated, stressed and enriched simultaneously E. coli O157:H7 (ATCC:43895) S. enterica (ATCC:13076) L. monocytogenes (ATCC: 13932) Low-level Inoculation: 20 replicates of 1 CFU/25g per matrix 1 CFU inoculation is AOAC-required lower limit of detection Inoculated into 1500 g matrix High-level Inoculation: 5 replicates of 5 CFU/25g per matrix 5 CFU inoculation is AOAC-required high level of detection Inoculated into 500 g matrix
4 Experimental Design Recovery and Detection Experimental Design After 48 hours, samples enriched at 37 C for 24 hours Enriched in PolySkope proprietary enrichment media (PMEM) Aerobic plate count (APC) performed on uninoculated control to determine native bacteria per gram Proprietary lysis procedure Maximizes recovery and detection of bacterial DNA PolySkope 1.0 assay run on lysate Real Time PCR detection of nucleic acid targets Run on ThermoFisher ABI QuantStudio 5 (96-well format)
5 AOAC Method Comparison Study Experimental Design Low-level Inoculation: 20 replicates of 1 CFU/25g deli turkey 1 CFU inoculation is AOAC-required lower limit of detection All 3 target organisms inoculated and incubated simultaneously 0.68 CFU/25g E. coli O157:H7 (ATCC:43895) 0.86 CFU/25g S. enterica (ATCC:13076) 1.1 CFU/25g L. monocytogenes (ATCC: 13932) High Level Inoculation: 5 replicates of 5 CFU/25g deli turkey All 3 target organisms inoculated and incubated simultaneously 3.42 CFU/25g E. coli O157:H7 (ATCC:43895) 4.3 CFU/25g S. enterica (ATCC:13076) 5.5 CFU/25g L. monocytogenes (ATCC: 13932) All bacteria inoculated into turkey and stored at 4 C for 48 hours As per AOAC guidelines 5
6 FDA BAM and USDA MLG Method Comparisons Study was carried out simultaneously using FDA BAM and USDA MLG reference methods E. coli O157 STEC method comparison (FDA BAM 4A) 25 grams deli turkey enriched in 225 ml Buffered Peptone Water with pyruvate (BPWp) at 37 C for 24 hours After 24 hours, replicates were streaked on Biolog Rainbow chromogenic agar and incubated at 37ºC for 24 hours S. enterica method comparison (USDA MLG 4.08) 25 grams deli turkey enriched in 225 ml Buffered Peptone Water (BPW) at 37 C for 24 hours After 24 hours, replicates were streaked on Hektoen chromogenic agar and incubated at 37ºC for 24 hours L. monocytogenes method comparison (USDA MLG 8.09) 25 grams deli turkey enriched in 225 ml University of Vermont modified listeria enrichment broth (UVM) at 30 C for 24 hours After 24 hours, replicates were streaked on Modified Oxford Medium (MOX) chromogenic plates and incubated at 35ºC for 24 hours. Additionally, supernatant from each enrichment was added to 10 ml of Fraser Broth (with supplements) and incubated at 35ºC for 24 hours as an additional confirmation test 6
7 AOAC Method Comparison Study Deli Turkey 1 CFU/25g
8 AOAC Method Comparison Validation 1 CFU E. coli O157:H7 STEC STX-1 and STX-2 Polyskope 1.0 detected STX-1 and STX-2 targets in 6/20 replicates (30% recovery) 8
9 AOAC Method Comparison Validation 1 CFU E. coli O157:H7 STEC EAE target Polyskope 1.0 detected E. coli EAE in 6/20 replicates (30% recovery)
10 AOAC Method Comparison Validation 1 CFU L. monocytogenes target Polyskope 1.0 detected the L. monocytogenes target in 15/20 replicates (75% recovery) 10
11 AOAC Method Comparison Validation 1 CFU S. enterica target Polyskope 1.0 detected the S. enterica target in 8/20 replicates (40% recovery) 11
12 All Targets in a Single Reaction 1 CFU Example of Polyskope 1.0 deteckng all three pathogens in a single enrichment and PCR reackon. 12
13 AOAC Method Comparison Validation 1 CFU Internal Control Polyskope 1.0 detected the internal control in 20/20 replicates (100% recovery)
14 Deli Turkey 1 CFU/25g Polyskope PCR Results STX-1/ Internal L. monocytogenes S. enterica EAE STX-2 Control Replicate "+/-" "+/-" "+/-" "+/-" "+/-"
15 Deli Turkey 1 CFU/25g AOAC BAM and MLG Method Comparison Results E. coli L. monocytogenes S. enterica Replicate "+/-" "+/-" "+/-"
16 Polyskope PCR and Method Comparison Results* - 1 CFU/25g Polyskope 1.0 % Positive USDA/FDA Methods % Positive STX-1/STX-2 and EAE targets 30% E. coli 35% L. monocytogenes target 75% L. monocytogenes 75% S. enterica target 40% S. enterica 35% *Please note that since this was conducted as an unpaired study, the posi4ve replicates will not be numerically matched when comparing the Polyskope PCR data to the BAM/MLG methods 16
17 Conclusion Polyskope PCR and Method Comparison Results - 1 CFU/25g Polyskope 1.0 PCR Results E. coli (STEC) targets (3 targets) STX-1/STX-2: Polyskope 1.0 identified 6/20 (30%) replicates as positive for STX-1/STX-2 EAE: Polyskope 1.0 identified 6/20 (30%) replicates as positive for EAE S. enterica (1 target): Polyskope 1.0 identified 8/20 (40%) replicates as positive for S. enterica. L. monocytogenes (1 target): Polyskope 1.0 identified 15/20 (75%) replicates as positive for L. monocytogenes Internal control detected on all replicates Methods Comparison Results E. coli O157: FDA BAM 4A identified 7/20 (35%) of replicates as positive for E. coli O157 S. enterica: USDA MLG 4.08 identified 7/20 (35%) of replicates as positive for S. enterica L. monocytogenes: USDA MLG 8.09 identified 15/20 (75%) of replicates as positive for L. monocytogenes 30-75% detec:on of all targets at 1 CFU/25g mee:ng AOAC requirements
18 AOAC Method Comparison Study Deli Turkey 5 CFU/25g
19 RTE Deli Meat (Deli Turkey) Validation (5 CFU/25g) Experimental Design High-level Inoculation: 5 replicates of 5 CFU/25g deli turkey 5 CFU inoculation is AOAC-required higher level of inoculation All 3 target organisms inoculated, stressed and enriched simultaneously 3.42 CFU/25g E. coli O157:H7 (ATCC:43895) 4.30 CFU/25g S. enterica (ATCC:13076) 5.50 CFU/25g L. monocytogenes (ATCC: 13932) All bacteria inoculated into deli turkey and stored at 4 C for 48 hours As per AOAC guidelines After 48 hour incubation, samples enriched at 37 C for 24 hours Enriched in PolySkope proprietary enrichment media (PMM) Aerobic plate count (APC) performed on uninoculated control indicated 2.7 X10^3 native bacteria per gram Proprietary lysis procedure Maximizes recovery and detection of bacterial DNA PolySkope 1.0 run on lysate (ThermoFisher Quantstudio 5 instrument) Maximizes recovery and detection of bacterial DNA
20 AOAC Method Comparison Validation 5 CFU E. coli O157:H7 STEC STX-1 and STX-2 Polyskope 1.0 detected STX-1 and STX-2 in 5/5 replicates (100% recovery)
21 AOAC Method Comparison Validation 5 CFU E. coli O157:H7 STEC EAE target Polyskope 1.0 detected E. coli EAE in 5/5 replicates (100% recovery)
22 AOAC Method Comparison Validation 5 CFU L. monocytogenes target Polyskope 1.0 detected L. monocytogenes in 5/5 replicates (100% recovery)
23 AOAC Method Comparison Validation 5 CFU S. enterica target Polyskope 1.0 detected S. enterica in 5/5 replicates (100% recovery)
24 AOAC Method Comparison Validation 5 CFU Internal Control Polyskope 1.0 detected the internal control in 5/5 replicates (100% recovery)
25 AOAC Method Comparison Validation 5 CFU All Targets Present Polyskope 1.0 easily detects all targets when present in the same reackon
26 Deli Turkey - 5 CFU/25g Polyskope PCR Results STX-1/ STX-2 L. monocytogenes S. enterica EAE Internal Control Replicate "+/-" "+/-" "+/-" "+/-" "+/-" Polyskope 1.0 detected all targets in 100% of replicates at a 5 CFU inoculakon level
27 Deli Turkey - 5 CFU/25g AOAC BAM and MLG Method Comparison Results E. coli L. monocytogenes S. enterica Replicate "+/-" "+/-" "+/-" BAM and MLG methods detected the 5 CFU inoculakon level in 100% of replicates for all pathogens
28 Polyskope PCR and Method Comparison Results - 5 CFU/25g Polyskope 1.0 % Positive USDA/FDA Methods % Positive STX-1/STX-2 and EAE targets 100% E. coli 100% L. monocytogenes target 100% L. monocytogenes 100% S. enterica target 100% S. enterica 100% 28
29 Conclusion Deli Turkey - 5 CFU/25g E. coli (STEC) targets (3 targets) STX-1/STX-2: Polyskope 1.0 identified 5/5 (100%) replicates as positive for STX-1/STX-2 EAE: Polyskope 1.0 identified 5/5 (100%) replicates as positive for EAE S. enterica (1 target): Polyskope 1.0 identified 5/5 (100%) replicates as positive for S. enterica. L. monocytogenes (1 target): Polyskope 1.0 identified 5/5 (100%) replicates as positive for L. monocytogenes Internal control detected in all replicates Methods Comparison Results E. coli O157: FDA BAM 4A identified 5/5 (100%) of replicates as positive for E. coli O157 S. enterica: USDA MLG 4.08 identified 5/5 (100%) of replicates as positive for S. enterica L. monocytogenes: USDA MLG 8.09 identified 5/5 (100%) of replicates as positive for L. monocytogenes 100% detec:on of all pathogens at 5 CFU/25g
30 AOAC Method Comparison Table of Results Method Agreement: Polyskope 1.0 vs Reference Methods Pathogen Innoculum Level MPN/25g Number of Samples PolySkope PosiKve Reference PosiKve Method Agreement SensiKvity Specificity L. mono Control % NA 100% High % 100% Low % 100% E. coli STEC Control % NA 100% High % 100% Low % 86% S. enterica Control % NA 100% High % 100% Low % 114% % method agreement in detec:on of all targets with sensi:vity and specificity mee:ng AOAC requirements 30
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