3/5/2018 Environmental Listeria Testing Common Issues: Results - PDF document

3/5/2018 Environmental Listeria Testing Common Issues: • Results come back too late to do anything meaningful with them. • Positives in Zone 1 / food contact surfaces must be reported so we don’t want to test there even though it could be critical for our process. Environmental Listeria Test Results in Less • It’s extremely difficult to perform vectoring Than One Hour after positives because by the time we get back results, we’ve cleaned at least once or twice. Environmental Listeria Testing Introducing Listeria Right Now Common Issues: Complete system for • We try to do as little Listeria testing as taking environmental possible since it could be traced back to Listeria tests with our facility via the retained culture. molecular-level accuracy • We’d like to hold product pending our that requires no Listeria testing results but it costs too enrichment and features a much in storage or our product has too total time-to-result of under short of a shelf life. one hour. System Description System Description • Isothermal Listeria Right Now Kit amplification system for 96 samples and reader (16 wells) • Environmental swabs • A computer with data for sample collection reporting software • Lysis buffer • Two heater blocks - components one at 37 o C and the other at 80 o C • ANSR reagents • Vortex Mixer (reaction tubes with internal positive control) Note: the positive control is not Listeria Vortex Mixer Heater blocks 1

3/5/2018 A Simple Procedure A Simple Procedure Swab a 4”X4” surface 4. Transfer .5ml solution to 1. 2. Express swab in cluster tube and incubate at 37 C for 10 minutes. Eppendorf tube w/ 1ml of lysis buffer 5. Transfer tubes to second 3. Vortex tube for several heating block at 80 C for 20 minutes seconds A Simple Procedure A Simple Procedure 6. Remove appropriate number 9. Vortex briefly and then return to of ANSR reagent tubes and reader place in reader for at least 10. Press Start on reader three minutes 7. Transfer 50 uL solution to reaction tube 8. Cap tubes A Simple Procedure Workflow Example: 7:00 a.m. Sample Collection 8:55 a.m. Read result. 11. Results will appear in 18 minutes 8:00 a.m. Samples arrive . Set-up – 4 – Relay to customer. 5 minutes 8:05 a.m. Express swab in 1 mL Lysis buffer. 2 – 3 8:37 a.m. minutes Transfer 50 uL to reaction tube and run on ANSR 8:07a.m . Incubate at 37 C reader - 18 for 10 minutes minutes 8:17 a.m. Incubate at 80 C for 20 minutes 8:56 a.m. Begin operations or re-clean and retest 2

3/5/2018 How is This Possible? How is This Possible? Neogen’s Listeria Right Now system is able to detect Ribosomal RNA is present in very low numbers of Listeria spp ., including L. much greater numbers in Listeria monocytogenes , from environmental samples without cells than a traditional DNA target (~1000 – 10,000 copies enrichment . • The system employs an isothermal, amplified nucleic per cell Vs 1 copy per cell for acid-based reaction to target rRNA. DNA). This can result in a 1,000 • Amplification occurs through a polymerization – 10,000 fold increase in target mechanism by a specific endonuclease. analyte concentration. • Detection occurs in real-time using a fluorescent, molecular beacon. How is This Possible? How is This Possible? The isothermal reaction within the Summary ANSR instrument produces a starting with significantly constant cycle of molecular more targets replication producing analyte copies much more quickly than traditional + a significantly faster cycle PCR reactions which run through a time series of heating and cooling = significantly faster results. cycles. Deep Dive - Isothermal Replication Deep Dive -Isothermal Replication • • A special primer The rRNA is released from the target cells within the reagent through the lysis tubes targets procedure specific regions of • A reverse the DNA and starts transcription reaction the amplification occurs converting rRNA to DNA process 3

3/5/2018 Deep Dive - Isothermal Replication Deep Dive - Isothermal Replication • An endonuclease, • Replicates one and nicking enzyme cleaves two repeat the the strand releasing the process rapidly and first replicate exponentially • Replicate one is bound creating millions of by a second primer and copies of the target. elongated using a polymerase and then cleaved by the nicking enzyme producing replicate two. Data – Internal Env. Surface Study Deep Dive - Detection Results and probability of detection calculations for the Listeria Right Now presumptive and culture confirmation assays • A special molecular beacon is part of Listeria Surface type Trial N LRN + Culture + the reagent mixture. CFU/swab 1 15 3 2 • If replicate two binds to the 2 15 10 7 Stainless steel Lm (4b) only molecular beacon, a quencher and 2438 5 5 5 0 5 0 0 fluorophore are spatially separated 1.8 20 8 7 Stainless steel Lm (4b) + background 1800 5 5 5 allowing the fluorescence to be 0 5 0 0 detected in the ANSR instrument 2.3 20 9 9 L. innocua + Plastic 2250 5 5 5 background 0 5 0 0 1.2 20 6 11 L. welshimeri + Sealed concrete 1550 5 5 5 background 0 5 0 0 1.93 20 14 9 Ceramic tile Lm (1/2a) + background 1930 5 5 5 0 5 0 0 Listeria Right Now has been validated on multiple environmental surfaces Data -Testing with Sanitizers Data - Limit Of Detection 120% Effects of Food Industry Sanitizers/Disinfectants on Organisms tested Listeria Right Now Assay L. monocytogenes 100% L. innocua Sample ID # swab LRN assays positive negative invalid L. welshimeri No Listeria organism on the surface 80% L. grayi % positive L. ivanovii 10% bleach 7 14 0 14 0 L. seeligeri Mandate Plus 7 14 0 14 0 60% Ster-Bact 7 14 14 0 0 Peroxyacetic acid 1% 4 4 0 4 0 L. mono, n=104 Peroxyacetic acid 2% 4 4 0 4 0 40% negative control 3 6 0 6 0 total (L. mono not included), n=200 With Listeria spp. on the surface 20% total (L. mono XY-12 4 8 8 0 0 included), n=304 Mandate Plus 4 8 8 0 0 0% Peroxyacetic acid 1% 4 4 4 0 0 ~1.5 CFU 2~ 3CFU ~4 CFU ~ 7-8 CFU ~16 CFU ~ 31 CFU Peroxyacetic acid 2% 4 4 4 0 0 Theoretical limit of detection = 2 CFU / swab Water control 4 8 1 7 0 95% Confidence Interval L.O.D. = 4 CFU / swab Conclusion: No interference from residual sanitizers 4

3/5/2018 Data – NSF International Survey Results: How would you use it? Table 4: Environmental surface study results for Listeria monocytogenes and background organisms on stainless steel. Food Safety professionals across Meat & Poultry, Dairy, Fresh Cut, Prepared Foods, Commercial Labs, Egg Processors, Ingredient Mfgr’s , Confectionary, Dessert Mfg’s , Flavors and Extracts, Canned Vegies, Tree Nuts, Grain Processing, Commercial Labs, Yogurt. With Listeria Right Now we could : o Use it as a process control. o Perform a corrective action much more quickly and fix “No false negatives, false positives or invalids were observed during an issue before it becomes a serious problem. this study. The data illustrates that under the conditions employed in o Perform investigations following a positive in near real- this study Listeria Right Now is as sensitive as the enrichment-based culture reference method for detection of L. monocytogenes on a time. stainless steel surface .” Survey Results: How would you use it? Early Feedback from Users With Listeria Right Now we could : With Listeria Right Now we can : o Perform vectoring, the process of tracing the sources and • Use it like an “ATP test for Listeria”. contamination paths of a pathogen, more successfully. • Commission new equipment much more quickly o Expand testing to include zone 1 areas - food contact – it used to take three days to move new surfaces which are commonly not sampled for Listeria equipment into production. Now it can be done due to the dire consequences of a positive. With LRN, we within a single shift. could find a positive on a zone 1 surface and immediately re-clean and retest to demonstrate the remedial action. • Inspect and validate areas following o Be more flexible and proactive with our sampling construction. program. A faster time to result expands the usefulness of • Test tools and other maintenance items to the system. minimize cross-contamination. National Vegetable Dip Company National Vegetable Dip Company • FDA did “swab -a- thon” no positives on product With flexibility of LRN system: • but found environmental positives. Rerouted traffic patterns for product and • Recall was ordered. personnel based on Listeria hot spots • Global food safety director decided to “eradicate • Uncovered previously unknown harborage sites Listeria from our facility”. “Listeria hunt”. like equipment feet and cable bundles because • of LRN’s swabs Reduced testing turnaround time from 48 or 72 • hours to one hour Refined cleaning practices and instituted twice- • With LRN, heavy cleaning, new practices, have daily cleaning for newly found harborage sites • reduced Listeria positive incidence from ~10% to Replaced rubber feet with stainless steel on <.5%. some equipment. 5