Validation of the PhageDx™ Listeria Assay for Detection of Listeria Spp. on Stainless Steel and Ceramic Environmental Surfaces AOAC Performance Tested MethodSM 102005.

BACKGROUND: The PhageDx™ Listeria Assay is a simple, specific, and sensitive assay based on the infection of Listeria spp. by selected bacteriophages and the resultant expression of a luciferase reporter gene. Results are generated in as little as 24.5 h for stainless steel and ceramic environmental surfaces.
OBJECTIVE: An AOAC Performance Tested MethodsSM (PTM) study was conducted to validate the PhageDx Listeria Assay for the detection of Listeria on stainless steel and ceramic surfaces.
METHOD: The performance of the PhageDx method was compared to that of the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM) Ch. 10. Inclusivity/exclusivity, product consistency and stability, and robustness testing also were conducted.
RESULTS: Inclusivity testing demonstrated that the reporter bacteriophages were specific for Listeria ssp. and detected 58/61 Listeria strains tested, including all 34 L. monocytogenes strains. The reporter bacteriophage also was shown to not detect 46/47 non-Listeria bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least 6 months. Matrix studies on stainless steel and ceramic surfaces showed that there was no significant difference between the PhageDx Listeria Assay and the FDA/BAM Chapter 10 reference method. CONCLUSIONS AND HIGHLIGHTS: The validation study demonstrates that the PhageDx Listeria Assay is an effective method for the detection of Listeria spp. on stainless steel and ceramic environmental surfaces and meets the qualifications for AOAC PTM status. © AOAC INTERNATIONAL 2021.

General Information

Listeria monocytogenes is a bacterial pathogen that can be found in moist environments such as soil, water, and decaying vegetation and when ingested, most commonly through eating contaminated food, can cause listeriosis. In the United States, listeriosis is estimated to affect approximately 1600 people resulting in about 260 deaths annually. Symptoms of infection include diarrhea, vomiting, nausea, headaches, stiff neck, confusion, loss of balance, convulsions, fever, and muscle aches. Populations that are particularly susceptible to the effects of listeriosis are pregnant women and their newborns, older adults (65+ years) and people with weakened immunity, where infections can result in death. Infections in pregnant women can also result in miscarriage, stillbirth, premature delivery, or life threatening infant infections (3, 4).

Common food sources of Listeria contamination include deli meats, dairy products, and produce. Listeria spp. can survive in a wide range of conditions typically used in food preservation such as extremes in temperature and pH and high salt concentrations. Food contamination commonly occurs via transfer from contaminated surfaces that the food comes in contact with (3, 4). Thus, an essential measure to prevent Listeria contamination is to monitor sanitation of food preparation areas.

The PhageDx™ Listeria Assay provides all the reagents necessary for sample infection, lysis, and luciferase detection. After a 20–24 h enrichment of the test portions in buffered Listeria enrichment broth (BLEB) at 35 ± 1°C for stainless steel and ceramic surfaces (4 × 4” test area), a portion of the enriched sample is placed in a well of a 96-well break-away plate and infected with the PhageDx Listeria Recombinant Phage at 30°C for 4 h. Then, lysis/luciferase substrate master mix is added to the infected sample and read on a luminometer for signal measurement. Results are expressed as relative light units (RLU). Total sample handling time is approximately 30 min.

Principle

The PhageDx Listeria Assay is a simple, specific, and sensitive diagnostic test for Listeria on ceramic and stainless-steel environmental surfaces. The kit is designed to be used with a Promega luminometer and the clear results indicate which samples contain viable Listeria. The assay consists of five basic steps; sample collection, enrichment, phage infection, substrate addition, and signal read. Presumptive results are available in 24.5 h which includes a 20–24 h enrichment step and a 4 h infection step. The PhageDx Listeria Assay is designed to be performed by qualified laboratory personnel in laboratories performing microbiological analysis.

The PhageDx Listeria Assay is based on the infection of Listeria spp. by bacteriophages and replication of the infecting bacteriophages within their specific hosts. Bacteriophages demonstrate a high specificity for their bacterial host and are capable of replicating within their host quickly to high numbers. The recombinant phages used in the PhageDx Listeria Assay also express a luciferase reporter during replication. The presence of Listeria spp. is determined by incubating the lysate with the appropriate luciferase substrate and detecting emitted light in a luminometer. An absence of detected light above background indicates that no viable Listeria are present in that sample.

Target organism.—Listeria spp. [L. monocytogenes (1/2a, 1/2b, 1/2c, 3a, 4a, 4b, 4c, 4d, 4e), L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, L. grayi].

Matrix.—Stainless-steel and ceramic surfaces (4 × 4”).

Summary of validated performance claims.—Performance comparable to that of the US Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM) Chapter 10, Detection of Listeria monocytogenes in Foods and Environmental Samples, and Enumeration of Listeria monocytogenes in Foods (1).

Probability of detection (POD).—The proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. POD is concentration dependent. Several POD measures can be calculated: PODR (reference method POD), PODC (confirmed candidate method POD), PODCP (candidate method presumptive result POD), and PODCC (candidate method confirmation result POD).

Difference of probabilities of detection (dPOD).—Difference of probabilities of detection is the difference between any two POD values. If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level (2).

Kit name.—PhageDx Listeria Assay.

Cat. No.—5013.

Ordering information.—Not applicable. For internal use at Laboratory Corporation of America only.

PhageDx Listeria recombinant phage.—Part No. 3401, 12 tubes containing 100 µL phage solution.

Lysis buffer.—Part No. 3010, 12 tubes containing 150 µL lysis buffer.

Assay buffer.—Part No. 3003, 12 tubes containing 500 µL assay buffer.

Luciferase substrate.—Part No. 3004, 12 tubes containing 10 µL luciferase substrate.

96-well break-apart plate.—Part No. 3005, one pouch containing white break-apart plate (8 wells × 12 strips).

Plate sealing tape.—Part No. 3011, 2 pieces per kit.

One package insert.—Part No. 3402.

Sample sponge/bag.—Polyurethane sponge with Letheen, EZ Reach™ Polyurethane Sponge Sampler with Letheen (World Bioproducts, cat. No. EZ18FR-10LET-PUR).

Racks for holding sample bag.

Buffered Listeria enrichment broth (BLEB).—(Thermo Scientific Remel™ Listeria Enrichment Broth, cat. No. R453692).

Sterile filtered tips.—For sampling and delivering of 10–1000 µL.

Appropriate personal protective equipment.—Seehttps://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2009-P.PDF (5). For confirmation (optional):

RAPID’ Listeria plates.—Bio-Rad RAPID’ L. mono Agar, cat. No. 3564293; RAPID’ L. mono supplement 1, cat. No. 3564294; RAPID’ L. mono supplement 2, Cat. No. 3564746.

Adjustable single channel pipettors.—For sample collection and delivery (10–1000 µL).

Vortex mixer.

Air incubators capable of 30 ± 1°C, 35 ± 1°C, and 37 ± 1°C.

Promega GloMax

Personal computer for luminometer control and data analysis.

The PhageDx Listeria Assay involves the enrichment of samples which may contain human pathogenic Listeria and have the potential for contamination with subsequent handling of those samples. This method should be conducted by properly trained laboratory personnel in a suitable microbiology laboratory in accordance with “Biosafety in Microbiological and Biomedical Laboratories,” US Department of Health and Human Services, https://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2009-P.PDF (5). Care should be taken when handling the sample and reagents while performing the method.

Materials and reagents provided in the PhageDx Listeria Assay are not considered hazardous if used according to the assay method. Please review the Material Safety Data Sheet prior to performing the assay.

Follow all relevant guidelines and laboratory protocols while performing the assay and manufacturer’s equipment instructions.

Prepare BLEB media according to manufacturer’s instructions.

Before using the reagents, flick or spin the tube to collect all of the solution at the bottom of the tube.

Before adding the pre-warmed BLEB to the sample, confirm that the media and incubator are warmed to 35 ± 1°C. Do not allow the pre-warmed media to cool before adding to the sample. Maintain the media at 35 ± 1°C in an incubator or water bath if preparing multiple samples. It is important to maintain the temperature of the sample and BLEB media used in the enrichment incubation.

Prepare the Promega luminometer by following the manufacturer’s cleaning procedure and make sure there are no signal “hot spots” that will affect the sample results by reading an empty plate. All signals should be less than 20 RLU. Set up the luminometer to read the appropriate wells on the plate using the pre-programmed PhageDx Assay protocol or, alternatively, set up a read protocol with a 3 min or 180 s delay between starting the program and the start of the signal reads and two plate read runs with a 1 s integration per sample.

For each surface type, use the EZ Reach Polyurethane Sponge Sampler to sample each test area. Swab environmental surfaces using firm and even pressure vertically (approximately 10 times), then flip the sampler and use the other side to swab horizontally (approximately 10 times) and diagonally (approximately 10 times). Replace sponge back into sample bag and break off and discard handle. Store at room temperature (20–25°C) for 2 h prior to enrichment.

Add 20 mL of BLEB media pre-warmed to 35 ± 1°C and massage sponge for 10–20 s. Incubate sponge at 35 ± 1°C for 20–24 h.

Remove the enriched samples from the incubator and massage the sponge in media for 10–20 s. Keeping the sponge in the bag, move sponge away from media and squeeze media from sponge and gently mix media in the bag. Note: It is critical that the analyte is released into the media and evenly distributed throughout the sample.

Immediately after mixing the media, using a single channel pipettor and fresh sterile tip for each sample, transfer 150 µL of the enriched sample into a well of the 96-well plate, one well per sample. Note: If continuing to confirmation assay, replace sponge into the media and continue to enrich at 35 ± 1°C for an additional 4–8 h for a total of 24–28 h.

Remove one tube containing the recombinant phage solution for each eight well strip used. Flick or spin the tube to collect all of the solution at the bottom of the tube. Add 10 µL of recombinant phage to the 150 µL sample and mix thoroughly by gently pipetting up and down, being careful not to introduce bubbles. Cover samples with plate sealing tape to prevent evaporation and cross contamination and place the 96-well plate in the 30 ± 1°C incubator for 4 h.

Remove one tube containing the lysis buffer, assay buffer, and substrate for each eight well strip used and thaw to room temperature. Flick or spin the tubes to collect all of the solution at the bottom of the tubes. Prepare the lysis/luciferase master mix by transferring the entire contents of assay buffer (0.5 mL) and lysis buffer (150 µL) tubes to the substrate tube (10 µL) and mix well by either vortexing or inverting the tube several times. Note: Use within 1 h of preparation.

Using a clean tip for each sample, add 65 µL of the lysis/luciferase master mix to each well using a single channel pipettor. Mix thoroughly by pipetting up and down, being careful not to introduce bubbles.

Once all of the samples have received the lysis/luciferase master mix, place the sample plate in the luminometer, close the lid, and initiate the read program.

The luminometer program will display the results on the screen as RLU values corresponding to the well positions of the break-apart plate.

Samples positive for Listeria will have a reading value of 300 RLU or greater. Negative samples will be less than 300 RLU.

Once all of the samples have been run and analyzed, remove the plate from the luminometer and follow the manufacturer’s instructions for cleaning the instrument and shut down. Note: In some cases, the PhageDx Listeria Assay will generate a very high signal and result in adjacent wells reading as a false positive due to a bleed-over signal from a well with a high signal. If a sample well is positive and has a 1000× lower signal than the adjacent sample well with a higher signal (typically in the range of 300–2000 RLU), this could be a result of bleed-over signal. In these cases, we recommend that the contents of the potential false positive well (lower RLU sample) be transferred to a new well at least a 2–3 well distance from the high signal well or to a new strip and re-read to confirm that the signal is from the sample and not a result of bleed-over signal.

Materials and Methods

Confirmation

We recommend that presumptive positives from the phage assay be confirmed.

To confirm, continue to enrich sponge samples for an additional 4–8 h for a total of 24–28 h at 35 ± 1°C. This is/can be done concurrently with the 4 h phage infection step. Sponge samples that were positive with the phage assay can then proceed to the next steps.

Massage sponge and contents of sample bag vigorously for 10–20 s to mix contents well. Remove a 100 µL aliquot and streak onto RAPID’ Listeria plates (Bio-Rad RAPID’ L. mono Agar, cat. No. 3564293; RAPID’ L. mono supplement 1, cat. No. 3564294; RAPID’ L. mono supplement 2, cat. No. 3564746). Incubate for an additional 24–48 h at 37 ± 1°C. L. monocytogenes forms blue (pale blue, grey-blue, or dark blue) colonies. L. ivanovii forms blue-green colonies with a yellow halo. Other Listeria spp. form white colonies with or without a yellow halo. Refer to manufacturer’s product insert for detailed description.

Confirm plate test results by testing individual colonies using approved qPCR tests. Alternatively, the user may use another approved reference method confirmation procedure.

Validation Study

This validation study was conducted under the AOAC Research Institute Performance Tested Method(s)SM (PTM) program and the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces, Appendix J (6). Method developer studies were conducted in the laboratories of Laboratory Corporation of America Holdings, and included the inclusivity/exclusivity study, product consistency and stability studies, and robustness testing. The independent laboratory study was conducted by Q Laboratories, Inc., and included inclusivity and exclusivity studies for selected strains and matrix studies for all claim matrixes.

Method Developer Studies

(a) Inclusivity and exclusivity studies.—

Inclusivity (Listeria) and exclusivity strains were obtained from academic, governmental, and commercially available sources. A total of 61 Listeria strains were tested for the inclusivity study, 54 strains by LabCorp and an additional seven by Q Laboratories. For inclusivity testing done at LabCorp, 54 Listeria species covering L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, 3a, 4a, 4b, 4c, 4d, and 4e, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi strains were examined. Each strain was grown at 35 ± 1°C for 20–24 h in BLEB medium. The cultures were then diluted to approximately 100 times the LOD50 of the PhageDx Listeria Assay and transferred to a 96-well plate.

A total of 47 non-Listeria strains were tested for the exclusivity study, 45 strains by LabCorp and three by Q Laboratories (Enterococcus faecalis ATCC 29212 tested by both LabCorp and Q Laboratories). For exclusivity testing done at LabCorp, 45 different strains of non-Listeria species and strains were grown under optimal enrichment conditions suitable to the strain type (medium and temperature) for a minimum of 20–24 h. One hundred microliters of the undiluted cultures were transferred to 96-well plates.

For both inclusivity and exclusivity studies, samples were infected with 10 µL of PhageDx Listeria phage reagent at 30 ± 1°C for 4 h. After infection, 65 µL of lysis/luciferase master mix was added to the wells and samples were read on a luminometer. Samples with a signal of ≥300 RLU were considered positive.

(b) Product consistency (lot-to-lot) and stability studies.—

Three separate production lots of PhageDx Listeria recombinant phage were prepared according to written manufacturing documents and tested according to quality control procedures. Quality control procedures verified that each lot when diluted to working concentration had the same titer, background, and level of detection. Recombinant phage reagents were aged between 0 and 6 months when assayed for stability.

Consistency and stability were done according to AOAC guidance, where a sample was inoculated with L. monocytogenes (ATCC 7302) to give fractional positives. Ten replicates were run in the PhageDx Assay, and the RLU values analyzed. A set of stability studies was also conducted using the non-target bacterium Enterococcus faecalis (ATCC 29212). Overnight cultures of E. faecalis were used directly in the assay.

(c) Robustness study.—

Three parameters were varied to demonstrate assay robustness: infection time (±30 m), recombinant phage concentration (±20%), and lysis/luciferase master mix amount (±5 µL). Briefly, stainless-steel surfaces were inoculated with L. monocytogenes (ATCC 19115) at a low level to generate partial positives or 100× E. faecalis (ATCC 29212) and allowed to dry and sit for 16–24 h at room temperature (20–25°C). The PhageDx Listeria Assay protocol was followed with the variations in infection time, recombinant phage concentration, and lysis/substrate master mix amounts as indicated in Table 4. Samples with RLU values of 300 or greater were considered positive. Samples were confirmed by allowing samples to enrich for a total of 24–28 h and plating on RAPID’ L. monocytogenes plates and incubating at 37 ± 1°C for an additional 24–48 h. The presence of blue (pale blue, grey-blue, or dark blue) colonies on selective plates indicated a positive result for L. monocytogenes.

Table 4.

Robustness study: impact of varying infection time, phage concentration, lysis/luciferase master mix concentration on PhageDx Listeria Assay results—POD comparison

Test conditiona	Test parameters			Nc	Test condition results				Nominal condition resultsb			dPODTNg	95% CIh
	Infectiontime, h	Volumephage,  µL	Volume substrate, µL		xd	PODTe	95% CI		x	PODNf	95% CI
Stainless steel surface—inoculated with L. monocytogenes ATCC 19115 (target) 55 CFU/test area
1	3.5	8	60	10	4	0.40	0.17, 0.69		4	0.40	0.17, 0.69	0.0	−0.25, 0.25
2	3.5	8	70	10	4	0.40	0.17, 0.69		4	0.40	0.17, 0.69	0.0	−0.25, 0.25
3	3.5	12	60	10	4	0.40	0.17, 0.69		4	0.40	0.17, 0.69	0.0	−0.25, 0.25
4	3.5	12	70	10	4	0.40	0.17, 0.69		4	0.40	0.17, 0.69	0.0	−0.25, 0.25
5	4.5	8	60	10	4	0.40	0.17, 0.69		4	0.40	0.17, 0.69	0.0	−0.25, 0.25
6	4.5	8	70	10	4	0.40	0.17, 0.69		4	0.40	0.17, 0.69	0.0	−0.25, 0.25
7	4.5	12	60	10	4	0.40	0.17, 0.69		4	0.40	0.17, 0.69	0.0	−0.25, 0.25
8	4.5	12	70	10	4	0.40	0.17, 0.69		4	0.40	0.17, 0.69	0.0	−0.25, 0.25
Stainless steel surface—inoculated with E. faecalis ATCC 29212 (non-target) 4950 CFU/test area
1	3.5	8	60	10	0	0.0	0.00, 0.28		0	0.0	0.00, 0.28	0.0	−0.25, 0.25
2	3.5	8	70	10	0	0.0	0.00, 0.28		0	0.0	0.00, 0.28	0.0	−0.25, 0.25
3	3.5	12	60	10	0	0.0	0.00, 0.28		0	0.0	0.00, 0.28	0.0	−0.25, 0.25
4	3.5	12	70	10	0	0.0	0.00, 0.28		0	0.0	0.00, 0.28	0.0	−0.25, 0.25
5	4.5	8	60	10	0	0.0	0.00, 0.28		0	0.0	0.00, 0.28	0.0	−0.25, 0.25
6	4.5	8	70	10	0	0.0	0.00, 0.28		0	0.0	0.00, 0.28	0.0	−0.25, 0.25
7	4.5	12	60	10	0	0.0	0.00, 0.28		0	0.0	0.00, 0.28	0.0	−0.25, 0.25
8	4.5	12	70	10	0	0.0	0.00, 0.28		0	0.0	0.00, 0.28	0.0	−0.25, 0.25

Each test condition is being compared to the nominal test condition.

Nominal condition = 4 h infection, 10 µL phage, 65 µL lysis/luciferase master mix substrate.

N = Number of test portions per condition.

x = Number of positive test portions per condition.

PODT = Positive outcomes divided by the total number of trials per condition.

PODN = Positive outcomes divided by the total number of trials per nominal condition.

dPODTN = Difference in POD between the test condition and nominal condition.

95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

Independent Laboratory Validation Study

(a) Inclusivity and exclusivity.—

The inclusivity and exclusivity study evaluated two strains of L. grayi, three strains of L. seeligeri, two strains of L. welshimeri, one strain of Enterococcus faecalis, one strain of Enterococcus faecium, and one strain of Streptococcus pyogenes. All cultures evaluated were propagated from a stock culture stored at −70°C to tryptic soy agar (TSA) with 5% sheep blood agar (SBA) and incubated for 24 ± 2 h at 35 ± 1°C. After incubation of the target organisms, a single colony from SBA was transferred to a 9 mL aliquot of BLEB and incubated for 20–24 h at 35 ± 1°C. Exclusivity strains were transferred to non-selective media and incubated under conditions for optimal growth. Inclusivity and exclusivity testing was done in the same manner as method developer’s inclusivity and exclusivity studies. All inclusivity and exclusivity strains were blind coded and randomized. Tests results were decoded and reported as either positive or negative.

(b) Matrix study.—

The independent laboratory evaluation included matrix studies for 4 × 4” test areas of stainless steel (18 GA 300 series, brush finish, NSF certified stainless steel) and ceramic (unglazed) surfaces, comparing the PhageDx Listeria Assay to the 2017 FDA/BAM Chapter 10. Within each sample set, there were five uninoculated samples (0 CFU/test portion), 20 low-level inoculated samples (0.2–2 CFU/test portion), and five high-level inoculated samples (2–10 CFU/test portion). The low inoculation level was designed to produce fractional positive results in which the candidate or reference method produced 5–15 positive results (25–75%).

All samples were analyzed by the PhageDx Listeria Assay following enrichment with pre-warmed (35 ± 1°C) BLEB and incubated for 20–24 h at 35 ± 1°C. Analysis was conducted after 20 h of enrichment. Regardless of presumptive results, all samples were culturally confirmed by the FDA/BAM Chapter 10 reference method. In addition, all samples were confirmed following the alternative confirmation as described in Confirmation. Final confirmation for all samples was obtained by Bruker MALDI Biotyper following AOAC Method 2017.10 (7).

(c) Organism preparation and inoculation.—

For ceramic surfaces, 4 × 4” areas were inoculated with 0.25 mL of diluted L. innocua (ATCC 43547) culture and sampled using sampling sponges pre-moistened in letheen broth. For stainless steel surfaces, 4 × 4” areas were inoculated with 0.25 mL of diluted L. monocytogenes 4a (CWD 1554). In addition to the L. monocytogenes culture, stainless-steel surface was co-inoculated with a competitor organism, Enterococcus faecalis (ATCC 29212), at 10× the level of the target organism. All cultures utilized were propagated from a stock culture stored at −70°C to SBA and incubated for 24 ± 2 h at 35 ± 1°C. After incubation, a single colony from SBA was transferred to a 9 mL aliquot of brain heart infusion (BHI) broth for 20–24 h at 35 ± 1°C. Each culture was then diluted to the target concentration by performing serial dilutions using 0.1% peptone water as the diluent. For the uninoculated test portions, sterile 0.1% peptone water was applied to the test area. Each surface was dried for 16–24 h at room temperature (24 ± 2°C) prior to sampling. To determine the inoculation level for the environmental surfaces, aliquots of each inoculum were plated onto TSA in triplicate.

(d) FDA/BAM Chapter 10.—

For environmental samples, sponges were pre-moistened with 10 mL of Dey-Engley (D/E) neutralizing broth. Following addition of D/E neutralizing broth surfaces were swabbed using firm and even pressure 10 times vertically and horizontally. All environmental samples were then stored at 22 ± 2°C for 2 h ± 15 min. Swabs were enriched with 90 mL of BLEB, massaged by hand for 2 min and incubated at 30 ± 1°C for 4 h. Following 4 h of incubation, selective supplements acriflavine (10 mg/L), sodium nalidixate (40 mg/L), and cycloheximide (50 mg/L) were added to each test portion and samples were re-incubated for 24–48 h.

After 24 h of total incubation, the enriched samples were streaked to modified Oxford (MOX) agar plates and incubated at 35 ± 1°C for 24–48 h. In addition to MOX each enriched sample was streaked to the chromogenic selective agar brilliance Listeria agar (BLA) and incubated at 37 ± 1°C for 24 ± 2 h. The enriched samples were re-incubated for an additional 24 h at 30 ± 1°C and then streaked to a second MOX and BLA agar plate which was incubated for 24–48 h at 35 ± 1°C and 37 ± 1°C for 24 ± 2 h, respectively. MOX and BLA agar plates were examined for suspect colonies, and if present, at least five colonies were streaked to TSA containing 0.6% yeast extract (TSA/YE). The TSA/YE plates were incubated at 35 ± 1°C for 24–48 h and then examined for purity. Pure colonies were tested for catalase reactivity and a Gram stain was conducted. A pure L. monocytogenes colony was transferred to trypticase soy broth containing 0.6% yeast extract (TSB/YE). The TSB/YE cultures were incubated at 25 ± 1°C overnight, or until the broth was turbid, indicating sufficient growth. Catalase-positive organisms were stabbed into SBA plates and incubated at 35 ± 1°C for 24–48 h. The TSB/YE tubes incubated at 25 ± 1°C were used to prepare a wet mount slide to determine motility pattern. After incubation, the SBA plates were examined for hemolysis. Final confirmation was conducted using Bruker MALDI Biotyper following AOAC Method 2017.10.

(e) PhageDx Listeria Assay.—

Sponges from ceramic (4 × 4” test area) and stainless steel (4 × 4” test area) swabbed surfaces were enriched according to the protocol described in Organism preparation and inoculation.

PhageDx Listeria Assay was performed as described in Sample preparation. Briefly, using a polyurethane sponge pre-moistened with letheen, surfaces were swabbed and the sponge placed into the sample bag and held at 22 ± 2°C for 2 h ± 15 m. Swabs were enriched with 20 mL of pre-warmed BLEB, massaged by hand for 10–20 s and incubated at 35 ± 1°C for 20 h. Following enrichment, 150 µL sample aliquots were transferred to a 96-well plate. Ten microliters of phage reagent was added to each sample and samples were incubated at 30 ± 1°C for 4 h. After infection, 65 µL of lysis/luciferase substrate master mix was added to the samples and then read on a luminometer. All samples were culturally confirmed by the FDA/BAM Chapter 10 reference method. All samples were also confirmed by an alternate confirmation described previously in Confirmation. Final confirmation for all samples was obtained by Bruker MALDI Biotyper following AOAC Method 2017.09.

Results

Inclusivity studies for the PhageDx Listeria Assay demonstrated that of the 61 Listeria inclusivity strains tested, 58 were detected and three were not detected (Table 1). All 34 L. monocytogenes strains tested were detected, while those strains that were not detected included one strain each of L. innocua (ATCC BAA-680), L. seeligeri (ATCC 51335), and L. welshimeri (ATCC 43551; Table 1). Those strains that were not detected can be divided into two categories. The first category includes those that would be detected with higher cell numbers. L. innocua (ATCC BAA-680) and L. welshimeri (ATCC 43551) were detected with 20 h enrichment, or within the recommended enrichment time frame for environmental samples (data not shown). L. seeligeri (ATCC 51335), however, remained negative with extended enrichment times.

Table 1.

PhageDx Listeria inclusivity list

No.	Species	Serogroupa	Source	Origin	PhageDx Result
1	L. monocytogenes	1/2a	ATCC 15313b	Rabbit	Positive
2	L. monocytogenes	1/2a	ATCC 51774	Human	Positive
3	L. monocytogenes	1/2a	ATCC 35152	Guinea Pig	Positive
4	L. monocytogenes	1/2a	ATCC BAA-679	Rabbit	Positive
5	L. monocytogenes	1/2a	ATCC 51775	Cheese	Positive
6	L. monocytogenes	1/2a	ATCC 19111	Poultry	Positive
7	L. monocytogenes	1/2a	ATCC 51772	Cheese	Positive
8	L. monocytogenes	1/2b	ATCC BAA-751	Not available	Positive
9	L. monocytogenes	1/2b	ATCC BAA-839	Not available	Positive
10	L. monocytogenes	1/2b	ATCC 51780	Cheese	Positive
11	L. monocytogenes	1/2c	ATCC 51779	Cheese	Positive
12	L. monocytogenes	2	ATCC 9525	Not available	Positive
13	L. monocytogenes	1/2C	ATCC 19112	Human	Positive
14	L. monocytogenes	3a	ATCC 19113	Human	Positive
15	L. monocytogenes	3a	ATCC 51782	Cheese	Positive
16	L. monocytogenes	4	ATCC 51781	Not available	Positive
17	L. monocytogenes	4a	ATCC 19114	Sheep	Positive
18	L. monocytogenes	4b	ATCC 19115	Human	Positive
19	L. monocytogenes	4b	Cornell FSL-J1-225c	Not available	Positive
20	L. monocytogenes	4b	ATCC 51776	Cheese	Positive
21	L. monocytogenes	4b	ATCC 51778	Cheese	Positive
22	L. monocytogenes	4b	ATCC 13932	Human	Positive
23	L. monocytogenes	4b	ATCC 51777	Cheese	Positive
24	L. monocytogenes	4c	ATCC 19116	Chicken	Positive
25	L. monocytogenes	4d	ATCC 19117	Sheep	Positive
26	L. monocytogenes	4e	ATCC 19118	Chicken	Positive
27	L. monocytogenes		ATCC 700402	Not available	Positive
28	L. monocytogenes		ATCC 7302	Not available	Positive
29	L. monocytogenes		ATCC 7644	Not available	Positive
30	L. monocytogenes		ATCC 23074	Not available	Positive
31	L. monocytogenes		ATCC 23073	Not available	Positive
32	L. monocytogenes		ATCC 43256	Not available	Positive
33	L. monocytogenes		ATCC 984	Not available	Positive
34	L. monocytogenes	4a	UVM CWD 1554d	Not available	Positive
35	L. grayi		ATCC 19120	Not available	Positive
36	L. grayi		ATCC 25401	Not available	Positive
37	L. grayi		ATCC 700545	Not available	Positive
38	L. grayie		QL 30911.12f	Environmental sample	Positive
39	L. grayi serovar Murrayie		NCTC10814g	Not available	Positive
40	L. innocua	6a	ATCC 33090	Not available	Positive
41	L. innocua		ATCC 51742	Not available	Positive
42	L. innocua	6b	ATCC 33091	Not available	Positive
43	L. innocua	6b	ATCC 43547	Not available	Positive
44	L. innocua	6a	ATCC BAA-680	Not available	Negativeh
45	L. ivanovii		ATCC 19119	Not available	Positive
46	L. ivanovii		ATCC 49953	Not available	Positive
47	L. ivanovii	5	ATCC BAA-678	Not available	Positive
48	L. ivanovii		ATCC BAA-753	Not available	Positive
49	L. ivanovii		ATCC 49954	Not available	Positive
50	L. seeligeri		ATCC 35967	Not available	Positive
51	L. seeligeri		ATCC 51334	Not available	Positive
52	L. seeligeri	4a	ATCC 51335	Not available	Negative
53	L. seeligerie		QL 031011.2	Creamer	Positive
54	L. seeligerie		QL 031011.5	Frozen vegetables	Positive
55	L. seeligerie		Cornell FSL-S4-035	Not available	Positive
56	L. welshimeri	6b	ATCC 35897	Not available	Positive
57	L. welshimeri	6a	ATCC 43551	Not available	Negativeh
58	L. welshimeri	6a	ATCC 43549	Not available	Positive
59	L. welshimeri	1/2b	ATCC 43550	Not available	Positive
60	L. welshimerie		QL 030911-(0).8	Beef	Positive
61	L. welshimerie		UVM LW001	Not available	Positive

Serogroup listed if applicable.

American Type Culture Collection, Manassas, VA.

Cornell University, Ithaca, NY.

University of Vermont, Burlington, VA.

Testing done by Q Laboratories.

Q Laboratories, Cincinnati, OH.

National Collection of Type Cultures, Proton Down, Salisbury, UK.

20 h enrichment generated positive result.

Exclusivity studies showed that of the 47 exclusivity strains examined, 46 of the 47 strains were negative (Table 2). One strain, Lactobacillus plantarum ATCC 14917, was reported as positive; however, the signal generated was significantly lower than that of Listeria strains (data not shown).

Table 2.

PhageDx Listeria exclusivity list

No.	Species	Source	PhageDx result
1	Acinetobacter baumannii	ATCC 19606a	Negative
2	A. calcoaceticus	ATCC 23055	Negative
3	Bacillus cereus	ATCC 14579	Negative
4	B. cereus	ATCC 13061	Negative
5	B. circulans	ATCC 61	Negative
6	B. coagulans	ATCC 7050	Negative
7	B. licheniformis	ATCC 9789	Negative
8	B. megaterium	ATCC 14581	Negative
9	B. mycoides	ATCC 6462	Negative
10	B. pumilus	ATCC 700814	Negative
11	B. subtilis	ATCC 23857	Negative
12	B. subtilis subsp. subtilis	ATCC 6051	Negative
13	B. weihenstephanensis	ATCC 12826	Negative
14	Citrobacter braakii	ATCC 51113	Negative
15	C. freundii	ATCC 8090	Negative
16	C. koseri	ATCC 25408	Negative
17	Cronobacter muytjensii	ATCC 51329	Negative
18	C. sakazakii	ATCC 12868	Negative
19	Escherichia coli	ATCC 9637	Negative
20	Edwardsiella tarda	ATCC 15947	Negative
21	Enterobacter aerogenes	ATCC 13048	Negative
22	E. cloacae, subsp cloacae	ATCC 13047	Negative
23	E. kobei	ATCC BAA-260	Negative
24	Enteroccus faecalis	ATCC 19433	Negative
25	E. faecalisb	ATCC 29212	Negative
26	E. faecium	ATCC 19434	Negative
27	E. faeciumc	ATCC 700221	Negative
28	Escherichia fergusonii	ATCC 35469	Negative
29	E. hermanni	ATCC 33650	Negative
30	Hafnia alevi	ATCC 13337	Negative
31	Klebsiella oxytoca	ATCC 43165	Negative
32	K. pneumonia	ATCC 4352	Negative
33	Lactobacillus plantarum	ATCC 14917	Positive
34	L. rhamnosus	ATCC 7469	Negative
35	Morganella morganii: subsp. Maorganii M11	ATCC 25830	Negative
36	Pluralibacter gergoviae	ATCC 33028	Negative
37	Proteus mirabilis	ATCC 43071	Negative
38	P. vulgaris	ATCC 33420	Negative
39	Pseudomonas aeruginosa; Strain Boston 41401	ATCC 27853	Negative
40	Serratia marcescens	ATCC 13880	Negative
41	Shigella flexneri	ATCC 12022	Negative
42	S. sonnei	ATCC 9290	Negative
43	Staphylococcus aureus	ATCC 27660	Negative
44	S. epidermidis	ATCC 14990	Negative
45	S. haemolyticus	ATCC 29970	Negative
46	Streptococcus pyogenesc	ATCC 19615	Negative
47	Yersinia enterocolitica	ATCC 23715	Negative

American Typle Culture Collection, Manassas, VA.

Testing done by both LabCorp and Q Laboratories.

Testing done by Q Laboratories, Cincinnati, OH.

Lot-to-lot studies showed that the PhageDx Listeria recombinant phages can be manufactured consistently and are stable for at least 6 months when stored at 4°C. Manufactured lots were made on 2/20/20, 4/20/20, and 8/20/20 according to written manufacturing documents. Each lot produced similar results when tested according to QC tests for bacteriophage concentration, background signal, and LOD. Performance tests to determine the stability of each lot were performed to determine the shelf life of the recombinant phage. These tests demonstrated that lots produced 0 months prior to testing showed no significant difference from lots produced at least 6 months prior to testing. Additionally, no variation in exclusivity was observed with these three recombinant phage lots in tests with E. faecalis. Results shown in Table 3.

Table 3.

Stability and consistency (lot-to-lot) of PhageDx Listeria recombinant phage—POD comparison

Phage lot No.	Lot age, months	Na	xb	PODAc	95% CI	Phage lot No.	Lot age, months	N	x	PODBd	95% CI	dPODABe	95% CIf
L. monocytogenes (target)
Bg	4	10	4	0.4	0.17, 0.69	Ch	0	10	6	0.6	0.31, 0.83	−0.20	−0.53, 0.21
Ai	6	10	4	0.4	0.17, 0.69	C	0	10	6	0.6	0.31, 0.83	−0.20	−0.53, 0.21
A	6	10	4	0.4	0.17, 0.69	B	4	10	4	0.4	0.17, 0.69	0.00	−0.37, 0.37
Enterococcus faecalis (non-target)
B	4	10	0	0.0	0.0, 0.28	C	0	10	0	0.0	0.0, 0.28	0.0	−0.28, 0.28
A	6	10	0	0.0	0.0, 0.28	C	0	10	0	0.0	0.0, 0.28	0.0	−0.28, 0.28
A	6	10	0	0.0	0.0, 0.28	B	4	10	0	0.0	0.0, 0.28	0.0	−0.28, 0.28

N = Number of test portions.

x = Number of positive test portions.

PODA = Positive outcomes divided by the total number of trials first member of pair.

PODB = Positive outcomes divided by the total number of trials second member of pair.

dPODAB = Difference in POD between the paired comparison.

95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

Lot B was produced 04/20/20.

Lot C was produced 08/20/20.

Lot A was produced 02/20/20.

Robustness testing of the PhageDx Listeria Assay demonstrated that variations in infection time, recombinant phage concentration, and lysis/luciferase master mix amounts do not alter the results compared to the standard protocol. Infection times of 3.5 and 4.5 h, recombinant phage volumes of 8 and 12 µL, and lysis/luciferase master mix volumes of 60 and 70 µL produced identical results to the standard protocol of 4 h infection, 10 µL of recombinant phage, and 65 µL of lysis/luciferase master mix in both uninoculated and low inoculum test samples (Table 4). These results indicate that these deviations from the PhageDx Listeria Assay protocol did not alter the final results.

Matrixes studies were performed as per criteria outlined in Appendix J of the Official Methods of Analysis Manual, and fractional positive results were obtained for the PhageDx Listeria Assay (6). For ceramic and stainless steel (4×4” test areas) environmental samples, the inoculum recovered for the target L. innocua and L. monocytogenes strains fell within the targeted levels, while the competitor organism was at a level at least 10 times higher. The probability of detection (POD) was calculated as the number of positive outcomes divided by the total number of trials (6). Comparisons of the PhageDx presumptive results to the confirmed results using the FDA/BAM Chapter 10 reference method showed no significant difference between the two (Table 5). No significant difference was found when comparing the results of the PhageDx presumptive results and FDA/BAM Chapter 10 results in an unpaired study (Table 6). There was also no significant difference in the PhageDx presumptive results and results of the PhageDx confirmation method (Table 7). Finally, there was no difference between the presumptive PhageDx results confirmed using the PhageDx confirmation method and the FDA/BAM reference method (Table 8). In summary, POD analysis between the PhageDx Listeria Assay method and the reference method indicated that there was no significant difference at the 5% level between the number of positive results by the two methods for both matrixes. The POD analysis between the PhageDx Listeria Assay presumptive and confirmed results indicated that there was no significant difference at the 5% level for the method for both matrixes. A summary of POD analyses (8) are presented in Tables 5–8.

Table 5.

PhageDx Listeria results: presumptive vs confirmed using the reference method procedure

Matrix	Strain	CFU/test area	Na	PhageDx Listeria presumptive			BAM Ch. 10 confirmed			dPODCPe	95% CIf
				xb	PODCPc	95% CI	x	PODCCd	95% CI
Stainless steel (4 × 4”)	L. monocytogenes 4a (CWD 1554)/E. faecalis ATCC 29212g,h	N/Ai	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	−0.47, 0.47
		48 & 510	20	8	0.40	0.22, 0.61	8	0.40	0.22, 0.61	0.00	−0.13, 0.13
		160 & 1800	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	−0.47, 0.47
Ceramic (4 × 4”)	L. innocua (ATCC 43547 6 b)	N/A	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	−0.47, 0.47
		60	20	9	0.45	0.26, 0.66	9	0.45	0.26, 0.66	0.00	−0.13, 0.13
		200	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	−0.47, 0.47

N = Number of test portions.

x = Number of positive test portions.

PODCP = Candidate method presumptive positive outcomes divided by the total number of trials.

PODCC = Candidate method confirmed positive outcomes (confirmed using the reference method procedure) divided by the total number of trials.

dPODCP = Difference between the candidate method presumptive result and candidate method confirmed result POD values.

95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

CWD = University of Vermont, Burlington, VT.

ATCC = American Type Culture Collection, Manassas, VA.

N/A = Not applicable.

Table 6.

Method comparison results: PhageDx Listeria vs BAM Ch. 10

Matrix	Strain	CFU/test area	Na	PhageDx Listeria results			BAM Ch. 10			dPODCe	95% CIf
				xb	PODCc	95% CI	x	PODRd	95% CI
Stainless steel (4 × 4”)	L. monocytogenes 4a (CWD 1554)/E. faecalis ATCC 29212g,h	N/Ai	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	−0.43, 0.43
		48 & 510	20	8	0.40	0.22, 0.61	8	0.40	0.22, 0.61	0.00	−0.28, 0.28
		160 & 1800	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	−0.43, 0.43
Ceramic (4 × 4”)	L. innocua (ATCC 43547 6 b)	N/A	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	−0.43, 0.43
		60	20	9	0.45	0.26, 0.66	7	0.35	0.18, 0.57	0.10	−0.19, 0.37
		200	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	−0.43, 0.43

N = Number of test portions.

x = Number of positive test portions.

PODC = Candidate method presumptive positive outcomes confirmed positive using the reference method procedure.

PODR = Reference method confirmed positive outcomes divided by the total number of trials.

dPODC = Difference between the candidate method and reference method POD values.

95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

CWD = University of Vermont, Burlington, VT.

ATCC = American Type Culture Collection, Manassas, VA.

N/A = Not applicable.

Table 7.

PhageDx Listeria results: presumptive vs confirmed using the PhageDx Listeria confirmation procedure

Matrix	Strain	CFU/test area	Na	PhageDx Listeria presumptive			PhageDx Listeria confirmed			dPODCPe	95% CIf
				xb	PODCPc	95% CI	x	PODCCd	95% CI
Stainless steel (4 × 4”)	L. monocytogenes 4a (CWD 1554)/E. faecalis ATCC 29212g,h	N/Ai	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	−0.47, 0.47
		48 & 510	20	8	0.40	0.22, 0.61	8	0.40	0.22, 0.61	0.00	−0.13, 0.13
		160 & 1800	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	−0.47, 0.47
Ceramic (4 × 4”)	L. innocua (ATCC 43547 6 b)	N/A	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	−0.47, 0.47
		60	20	9	0.45	0.26, 0.66	9	0.45	0.26, 0.66	0.00	−0.13, 0.13
		200	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	−0.47, 0.47

N = Number of test portions.

x = Number of positive test portions.

PODCP = Candidate method presumptive positive outcomes divided by the total number of trials.

PODCC = Candidate method confirmed positive outcomes (confirmed using the PhageDx Listeria recommended procedure) divided by the total number of trials.

dPODCP = Difference between the candidate method presumptive result and candidate method confirmed result POD values.

95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

CWD = University of Vermont, Burlington, VT.

ATCC = American Type Culture Collection, Manassas, VA.

N/A = Not applicable.

Table 8.

Method comparison results: PhageDx Listeria vs BAM Ch. 10

Matrix	Strain	CFU/test area	Na	PhageDx Listeria results			BAM Ch. 10			dPODCe	95% CIf
				xb	PODCc	95% CI	x	PODRd	95% CI
Stainless steel (4 × 4”)	L. monocytogenes 4a (CWD 1554)/E. faecalis ATCC 29212g,h	N/Ai	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	−0.47, 0.47
		48 & 510	20	8	0.40	0.22, 0.61	8	0.40	0.22, 0.61	0.00	−0.13, 0.13
		160 & 1800	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	−0.47, 0.47
Ceramic (4 × 4”)	L. innocua (ATCC 43547 6 b)	N/A	5	0	0.00	0.00, 0.43	0	0.00	0.00, 0.43	0.00	−0.43, 0.43
		60	20	9	0.45	0.26, 0.66	7	0.35	0.18, 0.57	0.10	−0.19, 0.37
		200	5	5	1.00	0.57, 1.00	5	1.00	0.57, 1.00	0.00	−0.43, 0.43

N = Number of test portions.

x = Number of positive test portions.

PODC = Candidate method presumptive positive outcomes confirmed positive using the PhageDx Listeria recommended procedure.

PODR = Reference method confirmed positive outcomes divided by the total number of trials.

dPODC = Difference between the candidate method and reference method POD values.

95% CI = If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level.

CWD = University of Vermont, Burlington, VT.

ATCC = American Type Culture Collection, Manassas, VA.

N/A = Not applicable.

Discussion

The results of this validation study show that the PhageDx Listeria Assay is an effective alternative to the FDA/BAM Chapter 10 for the detection of Listeria on stainless steel and ceramic surfaces.

In inclusivity and exclusivity testing, the method was shown to be specific for Listeria, correctly identifying 58/61 Listeria target strains including all 34 L. monocytogenes strains tested. The PhageDx Listeria Assay was unable to detect three strains within the inclusivity panel, one strain each of L. innocua (ATCC BAA-680), L. seeligeri (ATCC 51335), and L. welshimeri (ATCC 43551). It is unclear exactly why these strains failed to generate a positive result since the PhageDx Listeria Assay was able to detect other members of the species. One possible explanation is that these strains do not have the receptor(s) or the optimal receptors required for efficient recognition by the phage (9). Possessing a receptor that was sufficiently similar to Listeria also may explain why Lactobacillus plantarum generated a false positive result. Another possibility for a false negative may be that the strain has a mechanism that prevents the phage from producing luciferase. These mechanisms may include, but are not limited to, preventing phage absorption, preventing phage DNA entry, or the cutting of phage nucleic acids (10).

The recombinant phage can be produced consistently and is stable for 6 months when stored appropriately. Robustness testing of the PhageDx Listeria Assay indicated that the method works well when the assay parameters (infection time, recombinant phage concentration, and substrate amount) were varied from the stated protocol.

Independent laboratory testing demonstrated that the PhageDx Listeria Assay was able to detect Listeria at low levels and in the presence of competing bacteria on stainless steel and ceramic surfaces. These studies also demonstrated that the performance of the PhageDx Listeria Assay was not statistically different from that of FDA/BAM Chapter 10 for environmental surfaces. Additionally, the PhageDx confirmation procedure was shown to produce results identical to the FDA/BAM Chapter 10 reference method confirmation procedure.

The PhageDx Listeria Assay has a number of advantages over the FDA/BAM Chapter 10 reference method. In addition to being a specific assay, the results are easy to interpret as an RLU endpoint is used to determine the outcome of the assay. Another advantage is that PhageDx provides a presumptive positive result in as little as 24.5 h compared to 72+ h in the case of the FDA/BAM Chapter 10 reference method. PhageDx is also a simple test that involves only five basic steps: enrichment, sampling, infection, substrate addition, and signal readout. Finally, PhageDx Assay is a rapid method that offers a considerable time savings compared to the FDA/BAM Chapter 10 reference method.

Conclusion

Results of this validation study support the claim that the PhageDx Listeria Assay is a specific, sensitive, fast, and simple method for the detection of Listeria on stainless steel and ceramic surfaces and is statistically comparable to the FDA/BAM Chapter 10 reference method. By using a luciferase-expressing recombinant bacteriophage, the PhageDx Assay was able to detect a positive sample after 20 h enrichment and a 4 h infection for environmental surfaces. The PhageDx Listeria Assay thus offers a significantly shorter time to results compared with the FDA/BAM Chapter 10 reference method.