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

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Environmental Listeria Test Results in Less Than One Hour

Environmental Listeria Testing

• 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.

• It’s extremely difficult to perform vectoring

after positives because by the time we get back results, we’ve cleaned at least once or twice. Common Issues:

• We try to do as little Listeria testing as

possible since it could be traced back to

• ur facility via the retained culture.
• We’d like to hold product pending our

Listeria testing results but it costs too much in storage or our product has too short of a shelf life.

Environmental Listeria Testing

Common Issues: Introducing Listeria Right Now Complete system for taking environmental Listeria tests with molecular-level accuracy that requires no enrichment and features a total time-to-result of under

• ne hour.

System Description

Heater blocks

• Isothermal

amplification system and reader (16 wells)

• A computer with data

reporting software

• Two heater blocks -
• ne at 37oC and the
• ther at 80oC
• Vortex Mixer

Vortex Mixer

System Description

Listeria Right Now Kit for 96 samples

• Environmental swabs

for sample collection

• Lysis buffer

components

• ANSR reagents

(reaction tubes with internal positive control) Note: the positive control is not Listeria

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A Simple Procedure

1. Swab a 4”X4” surface 2. Express swab in Eppendorf tube w/ 1ml of lysis buffer 3. Vortex tube for several seconds

A Simple Procedure

4. Transfer .5ml solution to cluster tube and incubate at 37 C for 10 minutes. 5. Transfer tubes to second heating block at 80 C for 20 minutes

A Simple Procedure

6. Remove appropriate number

• f ANSR reagent tubes and

place in reader for at least three minutes 7. Transfer 50 uL solution to reaction tube 8. Cap tubes

A Simple Procedure

• 9. Vortex briefly and then return to

reader

• 10. Press Start on reader

A Simple Procedure

• 11. Results will appear in 18 minutes

Workflow Example: 7:00 a.m. Sample Collection

8:00 a.m. Samples arrive. Set-up – 4 – 5 minutes 8:05 a.m. Express swab in 1 mL Lysis buffer. 2 – 3 minutes 8:07a.m. Incubate at 37 C for 10 minutes 8:17 a.m. Incubate at 80 C for 20 minutes 8:37 a.m. Transfer 50 uL to reaction tube and run on ANSR reader - 18 minutes 8:55 a.m. Read result. Relay to customer.

8:56 a.m. Begin operations or re-clean and retest

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How is This Possible?

Neogen’s Listeria Right Now system is able to detect very low numbers of Listeria spp., including L. monocytogenes, from environmental samples without enrichment.

• The system employs an isothermal, amplified nucleic

acid-based reaction to target rRNA.

• Amplification occurs through a polymerization

mechanism by a specific endonuclease.

• Detection occurs in real-time using a fluorescent,

molecular beacon.

How is This Possible?

Ribosomal RNA is present in much greater numbers in Listeria cells than a traditional DNA target (~1000 – 10,000 copies per cell Vs 1 copy per cell for DNA). This can result in a 1,000 – 10,000 fold increase in target analyte concentration.

How is This Possible?

The isothermal reaction within the ANSR instrument produces a constant cycle of molecular replication producing analyte copies much more quickly than traditional PCR reactions which run through a series of heating and cooling cycles.

How is This Possible?

Summary

starting with significantly more targets + a significantly faster cycle time = significantly faster results.

Deep Dive - Isothermal Replication

• The rRNA is released

from the target cells through the lysis procedure

• A reverse

transcription reaction

• ccurs converting

rRNA to DNA

Deep Dive -Isothermal Replication

• A special primer

within the reagent tubes targets specific regions of the DNA and starts the amplification process

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Deep Dive - Isothermal Replication

• An endonuclease,

nicking enzyme cleaves the strand releasing the first replicate

• Replicate one is bound

by a second primer and elongated using a polymerase and then cleaved by the nicking enzyme producing replicate two.

Deep Dive - Isothermal Replication

• Replicates one and

two repeat the process rapidly and exponentially creating millions of copies of the target.

Deep Dive - Detection

• A special molecular beacon is part of

the reagent mixture.

• If replicate two binds to the

molecular beacon, a quencher and fluorophore are spatially separated allowing the fluorescence to be detected in the ANSR instrument

Data – Internal Env. Surface Study

Surface type Trial Listeria CFU/swab N LRN + Culture + Stainless steel Lm (4b) only 1 15 3 2 2 15 10 7 2438 5 5 5 5 Stainless steel Lm (4b) + background 1.8 20 8 7 1800 5 5 5 5 Plastic

• L. innocua +

background 2.3 20 9 9 2250 5 5 5 5 Sealed concrete

• L. welshimeri +

background 1.2 20 6 11 1550 5 5 5 5 Ceramic tile Lm (1/2a) + background 1.93 20 14 9 1930 5 5 5 5 Results and probability of detection calculations for the Listeria Right Now presumptive and culture confirmation assays

Listeria Right Now has been validated on multiple environmental surfaces

Data -Testing with Sanitizers

Effects of Food Industry Sanitizers/Disinfectants on Listeria Right Now Assay

Conclusion: No interference from residual sanitizers

Sample ID # swab LRN assays positive negative invalid No Listeria organism on the surface 10% bleach 7 14 14 Mandate Plus 7 14 14 Ster-Bact 7 14 14 Peroxyacetic acid 1% 4 4 4 Peroxyacetic acid 2% 4 4 4 negative control 3 6 6 With Listeria spp. on the surface XY-12 4 8 8 Mandate Plus 4 8 8 Peroxyacetic acid 1% 4 4 4 Peroxyacetic acid 2% 4 4 4 Water control 4 8 7 1

Data - Limit Of Detection

0% 20% 40% 60% 80% 100% 120% ~1.5 CFU 2~ 3CFU ~4 CFU ~ 7-8 CFU ~16 CFU ~ 31 CFU

% positive

• L. mono, n=104

total (L. mono not included), n=200 total (L. mono included), n=304

• L. monocytogenes
• L. innocua
• L. welshimeri
• L. grayi
• L. ivanovii
• L. seeligeri

Theoretical limit of detection = 2 CFU / swab 95% Confidence Interval L.O.D. = 4 CFU / swab

Organisms tested

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Data – NSF International

Table 4: Environmental surface study results for Listeria monocytogenes and background organisms on stainless steel.

“No false negatives, false positives or invalids were observed during this study. The data illustrates that under the conditions employed in this study Listeria Right Now is as sensitive as the enrichment-based culture reference method for detection of L. monocytogenes on a stainless steel surface.”

Survey Results: How would you use it?

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:

• Use it as a process control.
• Perform a corrective action much more quickly and fix

an issue before it becomes a serious problem.

• Perform investigations following a positive in near real-

time.

With Listeria Right Now we could:

• Perform vectoring, the process of tracing the sources and

contamination paths of a pathogen, more successfully.

• Expand testing to include zone 1 areas - food contact

surfaces which are commonly not sampled for Listeria due to the dire consequences of a positive. With LRN, we could find a positive on a zone 1 surface and immediately re-clean and retest to demonstrate the remedial action.

• Be more flexible and proactive with our sampling
• program. A faster time to result expands the usefulness of

the system.

Survey Results: How would you use it?

With Listeria Right Now we can:

• Use it like an “ATP test for Listeria”.
• Commission new equipment much more quickly

– it used to take three days to move new equipment into production. Now it can be done within a single shift.

• Inspect and validate areas following

construction.

• Test tools and other maintenance items to

minimize cross-contamination.

Early Feedback from Users

• FDA did “swab-a-thon” no positives on product

but found environmental positives.

• Recall was ordered.
• Global food safety director decided to “eradicate

Listeria from our facility”. “Listeria hunt”.

• Reduced testing turnaround time from 48 or 72

hours to one hour

• With LRN, heavy cleaning, new practices, have

reduced Listeria positive incidence from ~10% to <.5%.

National Vegetable Dip Company

With flexibility of LRN system:

• Rerouted traffic patterns for product and

personnel based on Listeria hot spots

• Uncovered previously unknown harborage sites

like equipment feet and cable bundles because

• f LRN’s swabs
• Refined cleaning practices and instituted twice-

daily cleaning for newly found harborage sites

• Replaced rubber feet with stainless steel on

some equipment.

National Vegetable Dip Company

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With flexibility of LRN system:

• Powder antimicrobials made cells non-viable

which could recover after hydration. Previous testing methods did not identify this issue. Now, “we don’t care about whether a cell is viable, we want to get rid of the regardless”.

• Can now test and hold
• State department of agriculture came in to find
• ut how they did it.

National Vegetable Dip Company Pricing

Heater blocks

Complete System: $14,000 96 Sample kit: $15.00 per sample

Questions

Thank you!