Validation of N-Light^TM  L. monocytogenes for Detection of Listeria monocytogenes on Environmental Surfaces: AOAC Performance Tested Method^SM 122002

Abstract

Background

The NEMIS Technologies N-Light^TM L. monocytogenes assay uses chemiluminescence designed for the qualitative detection of Listeria monocytogenes from environmental surface samples.

Objective

To validate the NEMIS Technologies N-Light L. monocytogenes assay as part of the AOAC Performance Tested Method^SM program for the detection of L. monocytogenes on stainless steel, plastic (polystyrene), and ceramic environmental surfaces.

Method

Using the Vitl Life Science Solutions Lu-mini luminometer, the NEMIS Technologies N-Light L. monocytogenes assay was compared to the ISO 11290–1:2017: Microbiology of the Food Chain—Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. – Part 1 using a 1" × 1" stainless steel test area in an unpaired study design.

Results

The NEMIS Technologies N-Light L. monocytogenes assay using the Vitl Life Science Solutions Lu-mini luminometer demonstrated no statistically significant differences between presumptive and confirmed results or between candidate and reference method results. Data for additional Performance Tested Method^SM studies met acceptance criteria requirements.

Conclusions

The NEMIS Technologies N-Light L. monocytogenes assay is an effective method for the qualitative detection of L. monocytogenes from stainless steel, plastic (polystyrene), and ceramic environmental surface samples.

Highlights

The NEMIS method is the first chemiluminescence detection system based on a novel, patented, dioxetane compound.

General Information

Listeriosis is a serious infection that is usually caused by Listeria monocytogenes. The bacteria are exclusively transmitted to humans after consumption of contaminated food. Hot dogs, dairy products, deli meats, and fresh produce are the primary sources for possible outbreaks. Food can be cross-contaminated due to improper hygiene conditions and insufficient cleaning and disinfection of environmental surfaces. The infection is most dangerous for pregnant women and their newborns, the elderly, or immunocompromised individuals and has a reported 1600 cases each year in the United States (1).

Principle

The NEMIS Technologies N-Light^TM L. monocytogenes assay uses synthetic molecules that are designed to detect live bacteria through chemiluminescence. The presence of L. monocytogenes is determined by the relative light unit (RLU) reading of the sample. If the sample eclipses the RLU threshold, which is set to 20 000 RLU, the sample is deemed positive for the presence of L. monocytogenes.

The procedure has a streamlined sampling protocol that requires no DNA purification or lysis process. After swabbing and incubating, the end user has to dispense the AquaSpark^TM tablet, which contains the chemiluminescent dioxetane molecule, wait 10 minutes, and insert the tube into the luminometer and will receive results in only 10 seconds.

Scope of Method

• Analyte.—Listeria monocytogenes.

• Matrixes.—Stainless steel [AISI 304 (1.4301), grade 2b finish], plastic (polystyrene), and ceramic (glazed earthen).

• Summary of validated performance claims.—The N-Light L. monocytogenes assay is equivalent in performance to the reference method ISO 11290-1:2017 for the detection of L. monocytogenes on environmental surfaces (stainless steel, plastic, and ceramic) after 24 hours of enrichment.

Definitions

• 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: POD_R (reference method POD), POD_C (confirmed candidate method POD), POD_CP (candidate method presumptive result POD), and POD_CC (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).

Materials and Methods

Test Kit Information

• Kit name.—N-Light L. monocytogenes.

Test Kit Components

• Swabs containing phosphate buffered saline (PBS).—200 swabs with 1 mL PBS.

• NEMIS Enrichment Broth L. mono.—200 × 2 mL tubes.

• Dispenser caps containing AquaSpark tablet.—200.

Additional Supplies and Reagents

• COPAN FLOQSwabs^®.

• Tube cutter.

• Vitl Life Science Solutions Lu-mini Luminometer.

Apparatus

• Dry bath incubator.—Capable of maintaining 37 ± 2°C.

• Vortex mixer.

• Serological pipette bulbs (Automatic Pipette).—For sampling and delivering of 1–10 mL.

• Serological pipettes.—Aerosol resistant.

• Refrigerator.—Capable of maintaining 2–8°C.

• Calibrated timer.

Cultures

• American Type Culture Collection (ATCC).—Manassas, VA.

• Collection de l’Institut Pasteur (CIP).—Paris, France.

• Biological Resource Center of institute Pasteur (CRBIP).—Paris, France.

• National Collection of Type Cultures (NCTC).—Salisbury, United Kingdom.

• Microbial Strain Collection (Nexidia).—Dijon, France.

Safety Precautions

The following general precautions should always be followed. Clean the workstations with the disinfectant of choice (e.g., sodium hypochlorite solution, phenol solution, quaternary ammonium solution) before and after use as part of aseptic techniques. In addition to cleaning workstations, work areas should be separated for the following: media preparation, sample preparation, and pathogen detection. Gloves and other personal protective equipment should be used at all times. The Vitl Life Science Solutions Lu-mini luminometer or supplies should never be touched without wearing gloves. Never reuse kit disposables and always change pipettes and pipette tips between samples.

• The NEMIS Technologies N-Light L. monocytogenes assay should be disposed of following procedures for infectious or potentially infectious products. User should wear appropriate personal protective equipment, including (but not limited to) protective disposable gloves, laboratory coats, and eye protection when handling samples and kit reagents. Wash hands thoroughly after handling specimens and reagents. It is the responsibility of each laboratory to handle waste and effluents produced according to their type and degree of hazardousness and to treat and dispose of them (or have them treated and disposed of) in accordance with local, state, and federal regulations. Strict compliance with BSL-2 practices should be followed.

• L. monocytogenes is a biosafety level 2 organism. Biological samples such as enrichments have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations on disposal of biological wastes. Wear appropriate protective equipment, which includes but is not limited to protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained in accordance with applicable regulatory and company/institution requirements before working with potentially infectious materials. All enrichment broths should be sterilized following confirmation.

General Preparation

• Use aseptic technique.

• Change pipette tips between samples.

• Do not reuse kit disposables.

• Clean workstations before and after use.

• Separate work areas for media preparation, sample preparation, and pathogen detection.

Sample Preparation

Surface areas such as stainless steel, plastic, and ceramic of 1" × 1" in size were sampled with a Butterfield’s phosphate buffer premoistened swab. After sampling, the swab is placed into a tube containing 2 mL of NEMIS enrichment broth L. monocytogenes and shaken vigorously for 10 s. The swab is then removed from the tube, and the cap containing the AquaSpark tablet is attached to the tube. The tube is then incubated at 37 ± 1°C for 24 ± 1 h in a dry heat block.

Analysis

N-Light method

• Remove samples from the incubator.

• Press button on the dispenser cap to release the AquaSpark tablet.

• Incubate for 10 min at ambient temperature (20–25°C).

• Read tube in the luminometer and obtain results.

Instrument Loading

• Open the lid to the instrument.

• Load sample tube into the instrument.

• Close the lid to the instrument.

• Press Start Run to initiate the run.

• When the run is completed, open the lid to the Lu-mini and remove the sample tube.

• Discard negative and positive controls as outlined in the proper laboratory procedure.

Data Analysis and Interpreting Results

• Viewing results.—

  1. Results are displayed after sample analysis.

  2. Sample analysis takes 10 seconds, and results are displayed automatically.

• Interpretation.—

  1. Samples with a reading of or above 20 000 RLU are considered positive.

Confirmation

The NEMIS Technologies N-Light L. monocytogenes assay test portions can be confirmed following the ISO 11290–1:2017 reference method for the detection of L. monocytogenes by streaking the samples onto Ottaviani Agosti Agar (OAA) and esculin-based media followed by incubation at 37°C for 24 h. Presumptive colonies may be further confirmed by standard biochemical and physiological tests (3).

Validation Study

The N-Light L. monocytogenes assay was validated according to the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces (2). The Performance Tested Methods^SM validation has two main parts: Independent Laboratory Validation Studies and Method Developer Studies. For this validation, the Method Developer Studies included an inclusivity/exclusivity study of the target microorganism (Listeria monocytogenes) and environmental surface studies (stainless steel, plastic, and ceramic). The Independent Laboratory Validation Study included a matrix study of one environmental surface (stainless steel).

Independent Laboratory Studies

• Methods.—The study was conducted according to the procedures outlined in the AOAC Research Institute Performance Tested Methods^SM program: Independent Laboratory Study for the Detection of Listeria monocytogenes on Environmental Surfaces (November 2019, Version 1) protocol (2). The NEMIS Technologies N-Light L. monocytogenes assay was compared to the ISO 11290-1:2017 reference method (3) by creating a total of 30 unpaired sample replicates for each method. Within each sample set, there were five uninoculated samples, 20 low-level inoculated samples, and five high-level inoculated samples following an unpaired study design. After sampling, swabs were incubated at 37 ± 1°C for 24 ± 1 h before being analyzed by the Lu-mini luminometer. The reference method swabs were evaluated at 30 ± 2°C after 24–26 h of enrichment. Regardless of the presumptive results for the method comparison, all samples were culturally confirmed following the ISO 11290-1:2017. Final confirmation for all samples was obtained by Bruker MALDI Biotyper following AOAC Method 2017.10 (4).

• Organism preparation and inoculation.—For stainless steel inoculation, a liquid culture of L. monocytogenes ATCC BAA-751 and Enterococcus faecalis ATCC 29212, as the competitor organism, were used for inoculation. Both cultures were propagated on tryptic soy agar with 5% sheep blood agar (SBA) from a stock culture stored at −70°C. The SBA was incubated for 24 ± 2 h at 35 ± 1°C. A single colony was transferred to brain heart infusion (BHI) broth and incubated for 24 ± 2 h at 35 ± 1°C.

  L. monocytogenes was diluted in BHI broth to a low level expected to yield fractional results and a high level expected to yield all positive results. E. faecalis was diluted in BHI broth to 10 times the concentration of the target organism on the stainless-steel surface. To determine the inoculation level of the environmental surfaces, aliquots of each inoculum were plated onto tryptic soy agar and incubated for 24 ± 2 h at 35 ± 1°C.

  The stainless-steel surface (1" × 1" test area) was inoculated with 0.1 mL of the diluted inoculum and allowed to dry for 16–24 h at room temperature (20–25°C) prior to sampling. For the uninoculated test portions, sterile BHI broth was used. The surfaces were sampled by premoistening a swab in Butterfield’s phosphate buffer. The surfaces were swabbed vertically approximately 10 times, and then the sampler was turned over and the other side was used to swab horizontally approximately 10 times and diagonally approximately 10 times.

  To detect L. monocytogenes from artificially contaminated stainless steel, ISO 11290–1:2017 was applied as the reference method (3). Swabs were premoistened in 1 mL of Dey/Engley (D/E) buffer. The surfaces were swabbed vertically approximately 10 times, and then the sampler was turned over and the other side was used to swab horizontally approximately 10 times and diagonally approximately 10 times. The swab was then placed back in the container and submerged in the D/E before being stored at room temperature (20–25°C) for 2 h ± 15 min. Swabs were then placed into a test tube containing 9 mL of half Fraser broth and incubated at 30 ± 1°C for 24–26 h. At 24 h of incubation, samples were streaked onto modified oxford agar (MOX) and Brilliance Listeria Agar (BLA) and incubated for 37 ± 1°C for 24 ± 2 h. From the same tubes, a 0.1 mL aliquot of the primary enrichment was added to 10 mL of Fraser broth (FB) and incubated at 37 ± 1°C for 24 ± 2 h. After 24 hours of incubation, samples were again streaked onto MOX and BLA and incubated for 37 ± 1°C for 24 ± 2 h. The FB tubes were reincubated for a total of 48 hours and were again streaked onto MOX and BLA and incubated for 37 ± 1°C for 24 ± 2 h. All agar plates were examined for suspect colonies, and if present, at least five colonies were streaked onto tryptic soy agar containing 0.6% yeast extract (TSA/YE). The TSA/YE plates were incubated at 37 ± 1°C for 18–24 h and then examined for purity. Pure colonies were tested for catalase reactivity and a gram stain was conducted. A pure Listeria colony was transferred to trypticase soy broth containing 0.6% yeast extract (TSB/YE). The TSB/YE cultures were incubated at 37 ± 1°C overnight, or until the broth was turbid, indicating sufficient growth. Catalase-positive organisms were streaked onto 5% SBA plates and incubated at 37 ± 1°C for 18–24 h. The TSB/YE tubes incubated at 37 ± 1°C were used to prepare a wet mount slide to determine motility pattern. After incubation, the SBA plates were examined for beta-hemolysis. Final confirmation was conducted using the Bruker MALDI Biotyper following AOAC Method 2017.10.

  For detection of L. monocytogenes from artificially contaminated stainless steel by the N-Light L. monocytogenes assay surfaces were inoculated and sampled according to the AOAC protocol. After incubation, samples were processed by the Lu-mini luminometer. Regardless of presumptive results, all samples were confirmed by the ISO 11290–1:2017 reference method. Final confirmations for all samples were obtained by the Bruker MALDI Biotyper following AOAC Method 2017.10 (4).

• Results.—As per criteria outlined in Appendix J of the Official Methods of Analysis Manual (5), fractional positive results were obtained for the NEMIS Technologies N-Light L. monocytogenes assay. The probability of detection (POD) was calculated as the number of positive outcomes divided by the total number of trials. The POD was calculated for the candidate presumptive results, POD_CP, the candidate confirmatory results, POD_CC, the difference in the candidate presumptive and confirmatory results, dPOD_CP, presumptive candidate results that confirmed positive, POD_C, the reference method, POD_R, and the difference in the confirmed candidate and reference methods, dPOD_C. The POD analysis between the NEMIS Technologies N-Light L. monocytogenes assay and the reference method indicated that there was no significant difference, with 95% confidence, between the number of positive results for the method. The POD analysis between the NEMIS Technologies N-Light L. monocytogenes assay presumptive and confirmed results indicated that there was no significant difference, with 95% confidence, for the method. A summary of POD analyses is presented in Tables 4 and 5 with the method developer matrix study data.

Table 1.

Inclusivity panel results

#	Organism	Serotype	Source	Strain	Origin	Results
1	Listeria monocytogenes	1/2a	ATCCa	15313	Rabbit	Positive
2	Listeria monocytogenes	1/2a	Nexidiab	NEX-636	Cheese	Positive
3	Listeria monocytogenes	1/2a	Nexidia	NEX-718	Food isolate	Positive
4	Listeria monocytogenes	1/2a	Nexidia	NEX-726	Food isolate	Positive
5	Listeria monocytogenes	1/2a	Nexidia	NEX-772	Cheese	Positive
6	Listeria monocytogenes	1/2a	Nexidia	NEX-773	Cheese	Positive
7	Listeria monocytogenes	1/2a	Nexidia	NEX-789	Cheese	Positive
8	Listeria monocytogenes	1/2a	Nexidia	NEX-865	Food isolate	Positive
9	Listeria monocytogenes	1/2a	Nexidia	NEX-866	Food isolate	Positive
10	Listeria monocytogenes	1/2a	Nexidia	NEX-939	Food isolate	Positive
11	Listeria monocytogenes	1/2a	Nexidia	NEX-952	Food isolate	Positive
12	Listeria monocytogenes	1/2a	Nexidia	NEX-1492	Food isolate	Positive
13	Listeria monocytogenes	1/2a	Nexidia	NEX-1494	Food isolate	Positive
14	Listeria monocytogenes	1/2a	Nexidia	NEX-1495	Food isolate	Positive
15	Listeria monocytogenes	1/2a	Nexidia	NEX-1497	Food isolate	Positive
16	Listeria monocytogenes	1/2a	Nexidia	NEX-1498	Food isolate	Positive
17	Listeria monocytogenes	1/2a	Nexidia	NEX-1506	Food isolate	Positive
18	Listeria monocytogenes	1/2b	CIPc	78.32 (= 105449)	Chinchilla	Positive
19	Listeria monocytogenes	1/2b	CIP	78.33 (= 105448)	Cerebrospinal fluid	Positive
20	Listeria monocytogenes	1/2b	Nexidia	NEX-626	Food isolate	Positive
21	Listeria monocytogenes	1/2b	Nexidia	NEX-648	Food isolate	Positive
22	Listeria monocytogenes	1/2b	Nexidia	NEX-720	Cheese	Positive
23	Listeria monocytogenes	1/2b	Nexidia	NEX-841	Food isolate	Positive
24	Listeria monocytogenes	1/2b	Nexidia	NEX-851	Food isolate	Positive
25	Listeria monocytogenes	1/2b	Nexidia	NEX-1493	Food isolate	Positive
26	Listeria monocytogenes	1/2b	Nexidia	NEX-870	Food isolate	Positive
27	Listeria monocytogenes	1/2b	Nexidia	BAA-751	–	Positive
28	Listeria monocytogenes	1/2c	Nexidia	NEX-777	Food isolate	Positive
29	Listeria monocytogenes	1/2c	Nexidia	NEX-869	Food isolate	Positive
30	Listeria monocytogenes	1/2c	Nexidia	NEX-868	Food isolate	Positive
31	Listeria monocytogenes	1/2c	ATCC	7644	Human	Positive
32	Listeria monocytogenes	3a	CIP	78.34	Human	Positive
33	Listeria monocytogenes	3a	Nexidia	NEX-627	Food isolate	Positive
34	Listeria monocytogenes	3a	Nexidia	NEX-1496	Food isolate	Positive
35	Listeria monocytogenes	3a	Nexidia	NEX-1505	Food isolate	Positive
36	Listeria monocytogenes	3b	CIP	78.35	Spinal fluid	Positive
37	Listeria monocytogenes	3b	CRBIPd	13.109	Environment	Positive
38	Listeria monocytogenes	3c	CIP	78.36	–	Positive
39	Listeria monocytogenes	3c	CRBIP	13.36	Meat product	Positive
40	Listeria monocytogenes	4a	NCTCe	5214	Brain sheep	Positive
41	Listeria monocytogenes	4b	CIP	78.39	Chicken	Positive
42	Listeria monocytogenes	4b	CRBIP	13.107	Environment	Positive
43	Listeria monocytogenes	4b	Nexidia	NEX-642	Cheese	Positive
44	Listeria monocytogenes	4b	Nexidia	NEX-754	Food isolate	Positive
45	Listeria monocytogenes	4b	Nexidia	NEX-929	Food isolate	Positive
46	Listeria monocytogenes	4b	Nexidia	NEX-1504	Food isolate	Positive
47	Listeria monocytogenes	4c	ATCC	19116	Chicken	Positive
48	Listeria monocytogenes	4d	ATCC	19118	Sheep	Positive
49	Listeria monocytogenes	4d	NCTC	10888	Sheep	Positive
50	Listeria monocytogenes	4e	CIP	78.41	Chicken	Positive
51	Listeria monocytogenes	7	Nexidia	NEX-843	Food isolate	Positive
52	Listeria monocytogenes	7	CIP	78.43	–	Positive

a

ATCC = American Type Culture Collection, Manassas, VA.

b

Nexidia = Microbial Strain Collection, Dijon, France.

c

CIP = Collection de l’Institut Pasteur, Paris, France.

d

CRBIP = Biological Resource Center of Institute Pasteur, Paris, France.

e

NCTC = National Collection of Type Cultures, Salisbury, UK.

Table 2.

Exclusivity panel results

#	Organism	Serotype	Source	Strain	Origin	Results
1	Listeria innocua	–	ATCCa	33090	Cow brain	Negative
2	Listeria innocua	–	Nexidiab	NEX-1849	Milk	Negative
3	Listeria ivanovii	–	ATCC	BAA-139	Washing water	Negative
4	Listeria ivanovii	–	Nexidia	NEX-923	Duck breast	Negative
5	Listeria grayi	–	CIPc	103321	Food	Negative
6	Listeria seeligeri	–	CIP	100100	Soil	Negative
7	Listeria seeligeri	–	Nexidia	NEX-1848	Milk	Negative
8	Listeria welshimerii	–	CIP	81.49	Decaying vegetation	Negative
9	Lactococcus lactis	–	ATCC	11454	–	Negative
10	Lactococcus lactis	–	Nexidia	NEX-363	–	Negative
11	Streptococcus thermophilus	–	Nexidia	NEX-371	Food isolate	Negative
12	Lactobacillus plantarum	–	ATCC	14917	Pickled cabbage	Negative
13	Lactobacillus plantarum	–	Nexidia	NEX-EC-EnrE8	Surface	Negative
14	Lactobacillus buchneri	–	Nexidia	NEX-EC-E8	Surface	Negative
15	Klebsiella pneumoniae	–	ATCC	4352	Dairy products: cow’s milk	Negative
16	Enterobacter amnigenus	–	Nexidia	NEX-764	Cocoa	Negative
17	Cronobacter sakazakii	–	ATCC	12868	–	Negative
18	Escherichia coli	–	ATCC	8739	Feces	Negative
19	Citrobacter braakii	–	ATCC	51113	Snake	Negative
20	Pantoea agglomerans	–	CIP	82.100	Corn crops	Negative
21	Salmonella Typhimurium	–	ATCC	14028	Chicken	Negative
22	Pseudomonas aeruginosa	–	ATCC	15442	Animal room water bottle	Negative
23	Enterococcus avium	–	ATCC	14025	–	Negative
24	Enterococcus faecalis	–	ATCC	51299	Peritoneal fluid	Negative
25	Enterococcus hirae	–	ATCC	10541	–	Negative
26	Staphylococcus aureus	–	Nexidia	NEX-1093	Raw milk cheese	Negative
27	Staphylococcus aureus	–	Nexidia	NEX-1069	Mayonnaise	Negative
28	Bacillus cereus	–	CIP	105151	–	Negative
29	Bacillus subtilis	–	ATCC	6051	–	Negative
30	Clostridium perfringens	–	ATCC	13124	–	Negative

a

ATCC = American Type Culture Collection, Manassas, VA.

b

Nexidia = Microbial Strain Collection, Dijon, France.

c

CIP = Collection de l’Institut Pasteur, Paris, France.

Table 3.

Matrix studies summary

Environmental surface	Inoculation organisms	Inoculation level		Replicates
Stainless steel	L. monocytogenes ATCC BAA-751	Negative control	0 cfu/area	5
		Low	50–80 cfu/area	20
		High	1 × 103–1 × 104 cfu/area	5
Stainless steel	L. monocytogenes ATCC BAA-751 10–100× E. faecalis ATCC 21292	Negative control	LM: 0 cfu/area EF: 1 × 103–1 × 104 cfu/area	5
		Low	LM: 1 × 102–5 × 102 cfu/area EF: 1 × 103–1 × 104 cfu/area	20
		High	LM: 1 × 102–1 × 104 cfu/area EF: 1 × 103–5 × 105 cfu/area	5
Plastic	L. monocytogenes ATCC 7644	Negative control	0 cfu/area	5
		Low	100–150 cfu/area	20
		High	1 × 103–1 × 104 cfu/area	5
Ceramic	L. monocytogenes ATCC 19118	Negative control	0 cfu/area	5
		Low	2 × 102–2.5 × 102 cfu/area	20
		High	1 × 104–1 × 105 cfu/area	5

Table 4.

NEMIS Technologies N-Light L. monocytogenes assay: presumptive versus confirmed

Matrix	Strain	CFU/test areaa	Nb	Candidate method presumptive			Candidate method confirmed			dPODCPf	95% CIg
				xc	PODCPd	95% CI	x	PODCCe	95% CI
Stainless steel	Listeria monocytogenes ATCCh BAA-751 (1/2b)	0	5	0	0.00	(0.00, 0.43)	0	0.00	(0.00, 0.43)	0.00	(−0.43, 0.43)
		72	20	19	0.95	(0.76, 0.99)	19	0.95	(0.76, 0.99)	0.00	(−0.13, 0.13)
		2 200	5	5	1.00	(0.57, 1.00)	5	1.00	(0.57, 1.00)	0.00	(−0.43, 0.43)
Stainless steeli	Listeria monocytogenes ATCC BAA-751 & Enterococcus faecalis ATCC 29212	0	5	0	0.00	(0.00, 0.43)	0	0.00	(0.00, 0.43)	0.00	(−0.47, 0.47)
		73 & 810	20	8	0.40	(0.22, 0.61)	8	0.40	(0.22, 0.61)	0.40	(−0.13, 0.13)
		220 & 2400	5	5	1.00	(0.57, 1.00)	5	1.00	(0.57, 1.00)	1.00	(−0.47, 0.47)
Plastic	Listeria monocytogenes ATCC 7644 (1/2c)	0	5	0	0.00	(0.00, 0.43)	0	0.00	(0.00, 0.43)	0.00	(−0.43, 0.43)
		115	20	11	0.55	(0.34, 0.74)	11	0.55	(0.34, 0.74)	0.00	(−0.13, 0.13)
		10 000	5	4	0.8	(0.38, 0.96)	4	0.80	(0.38, 0.96)	0.00	(−0.45, 0.45)
Ceramic	Listeria monocytogenes ATCC 19118 (4d)	0	5	0	0.00	(0.00, 0.43)	0	0.00	(0.00, 0.43)	0.00	(−0.43, 0.43)
		210	20	16	0.8	(0.58, 0.92)	16	0.8	(0.58, 0.92)	0.00	(−0.13, 0.13)
		22 000	5	5	1.00	(0.57, 1.00)	5	1.00	(0.57, 1.00)	0.00	(−0.43, 0.43)

a

CFU/test area determined by plating the inoculum in duplicate.

b

N = Number of test portions.

c

x = Number of positive test portions.

d

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

e

POD_CC = Candidate method confirmed positive outcomes divided by the total number of trials.

f

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

g

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

h

ATCC = American Type Culture Collection, Manassas, VA.

i

Performed by independent AOAC-certified laboratory Q Laboratories, Cincinnati, OH.

Table 5.

Method comparison results: NEMIS Technologies N-Light L. monocytogenes assay versus ISO 11290–1

Matrix	Strain	CFU/Test Areaa	Nb	Candidate method results (confirmed)			ISO 11290–1 results (confirmed)			dPODCf	95% CIg
				xc	PODCd	95% CI	x	PODRe	95% CI
Stainless steel	Listeria monocytogenes ATCCh BAA-751 (1/2b)	0	5	0	0.00	(0.00, 0.43)	0	0.00	(0.00, 0.43)	0.00	(−0.47, 0.47)
		72	20	19	0.95	(0.76, 0.99)	15	0.75	(0.53, 0.89)	0.20	(−0.03, 0.42)
		2200	5	5	1.00	(0.57, 1.00)	5	1.00	(0.57, 1.00)	0.00	(−0.47, 0.47)
Stainless steeli	Listeria monocytogenes ATCC BAA-751 & Enterococcus faecalis ATCC 29212	0	5	0	0.00	(0.00, 0.43)	0	0.00	(0.00, 0.43)	0.00	(−0.43, 0.43)
		73 & 810	20	8	0.4	(0.22, 0.61)	6	0.30	(0.15, 0.52)	0.10	(−0.18, 0.36)
		220 & 2400	5	5	1.00	(0.57, 1.00)	5	1.00	(0.57, 1.00)	0.00	(−0.43, 0.43)
Plastic	Listeria monocytogenes ATCC 7644 (1/2c)	0	5	0	0.00	(0.00, 0.43)	0	0.00	(0.00, 0.43)	0.00	(−0.47, 0.47)
		115	20	11	0.55	(0.34, 0.74)	14	0.70	(0.48, 0.85)	−0.15	(−0.41, 0.14)
		10 000	5	4	0.80	(0.38, 0.96)	5	1.00	(0.57, 1.00)	−0.20	(−0.62, 0.28)
Ceramic	Listeria monocytogenes ATCC 19118 (4d)	0	5	0	0.00	(0.00, 0.43)	0	0.00	(0.00, 0.43)	0.00	(−0.47, 0.47)
		210	20	16	0.80	(0.58, 0.92)	12	0.60	(0.39, 0.78)	0.20	(−0.08, 0.44)
		22 000	5	5	1.00	(0.57, 1.00)	4	0.8	(0.38, 0.96)	0.20	(−0.28,0.62)

a

CFU/Test area determined by plating the inoculum in triplicate.

b

N = Number of test portions.

c

x = Number of positive test portions.

d

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

e

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

f

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

g

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

h

ATCC = American Type Culture Collection, Manassas, VA.

i

Performed by independent AOAC-certified laboratory Q Laboratories, Cincinnati, OH.

Method Developer Studies

• Methods.—The inclusivity/exclusivity study examined the ability of the N-Light L. monocytogenes assay to detect a variety of the claimed target strains (L. monocytogenes) and to distinguish those strains from closely related nontarget strains and species. Fifty-two inclusivity strains, covering serotypes 1/2a, 1/2 b, 1/2c, 3a, 3 b, 3c, 4a, 4 b, 4c, 4d, 4e, and 7, were cultured in NEMIS enrichment media for 24 ± 1 h at 37 ± 1°C. Each obtained suspension was diluted 10 times in 2 mL of fresh NEMIS enrichment media to 100 times the LOD50 of the method. One replicate per strain was tested.

  Exclusivity strains included 30 different strains of closely related non-L. monocytogenes strains including other Listeria species strains (L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi). Exclusivity strains were cultivated in nonselective media (M17 broth, BHI broth, or MRS broth depending on bacterial species) at conditions for optimal growth. Exclusivity cultures were not diluted prior to analysis. After incubation AquaSpark tablets were released from the caps to the suspension. Tubes were then mixed by vortex for 30 seconds. After 10 min of incubation at room temperature (20–25°C), luminescence was determined (Lu-mini; Vitl).

• Results.—Of the 52 specific inclusivity strains tested, 52 were detected by the N-Light L. monocytogenes assay (Table 1). Of the 30 specific exclusivity strains tested, 30 were not detected by the N-Light L. monocytogenes assay (Table 2). OD_600nm measurement of each suspension before N-Light L. monocytogenes test confirmed that all strains have grown (data not shown).

Matrix Study

• Methods.—The N-Light L. monocytogenes assay was compared to the cultural reference method for detection of Listeria monocytogenes (ISO 11290–1:2017) on environmental surfaces. Three types of environmental surfaces were tested: stainless steel (AISI 304 (1.4301), grade 2 b finish, plastic (polystyrene), and ceramic (glazed earthen material).

  For each surface the study included five replicate test portions of uninoculated matrix, 20 replicate test portions at a low level to yield fractionally positive results, and five replicate test portions at a high level to yield consistently positive results. Fractionally positive results, those in which at least one of the methods (candidate or reference) yields 5–15 positive results out of 20 replicates examined for the low level of inoculation, are required for each matrix tested. This is an unpaired study. Separate test portions were prepared for the candidate method and each reference method.

  Three L. monocytogenes strains were used: ATCC BAA-751 (serotype 1/2 b), ATCC 19118 (serotype 4d), and ATCC 7644 (serotype 1/2c). For stainless steel, E. faecalis ATCC 21292 was used as the competitor organism. For pure inoculum preparation, the working suspension of each strain was diluted in fresh BHI broth to obtain the required concentration. Bacterial concentration of inoculum was adjusted by measurement of the optical density at 600 nm and was controlled by plating the inoculum in triplicate on BHI agar plates after serial decimal dilution if needed. After 48 h of incubation at 37 ± 2°C, colonies were counted on plates that presented between 15 and 300 colonies.

  For the matrix study with competitive flora, E. faecalis ATCC 21292 was co-inoculated with L. monocytogenes ATCC BAA-751 on the stainless-steel surface. The competitor organism was inoculated at 10–100 times the level of the target strain. For this, a mixed culture was prepared. The work cultures of L. monocytogenes ATCC BAA-751 and the E. faecalis ATCC 21292 were diluted in fresh BHI broth to obtain a concentration of about twice the target concentration. The two diluted cultures were then mixed (1:1) to obtain the inoculum. L. monocytogenes concentration in the mixed suspension was determined by plating the inoculum onto PALCAM plates in triplicate after serial decimal dilution. E. faecalis concentration in the mixed suspension was determined by plating the inoculum onto KAAA plates (Kanamycin Aesculin Azide Agar) base in triplicate after serial decimal dilution. After 24 to 48 h of incubation at 37 ± 2°C, colonies were counted on plates that presented between 15 and 300 colonies. For this experiment, a negative control was prepared by diluting the E. faecalis ATCC 21292 culture in fresh BHI broth to obtain the same concentration as in the low concentration of the mixed culture.

  For each type of surface, nine 1′ × 1′ areas were defined on 4.7′ × 4.7′ plates. Each plate was washed with a specific dishwashing liquid (Anios) and decontaminated in 70% (v/v) ethanol bath for 15 min. The plates were then removed from the bath and allowed to dry for at least one hour under the flow of a biosafety cabinet.

  Each 1′ × 1′ area was inoculated with 100 µL of adequate inoculum or sterile BHI broth (Table 3). Drops were spread using a sterile loop to distribute the inoculum evenly over the surface. For drying, surfaces were placed in closed Petri dishes (245 × 245 cm) and under laminar flow for 18 h. During drying, room temperature was kept at 22 ± 2°C.

  For the reference method, e.g., ISO 11290–1:2017 (3), premoistened swabs (CLASSIQSwab^TM—COPAN) with phosphate saline buffer (1×; pH 7.2 ± 0.2) were used. Environmental surfaces were swabbed using firm and even pressure vertically (approximately 10 times), and then the sampler flipped and the other side used to sample horizontally (approximately 10 times) and diagonally (approximately 10 times). Swabs were introduced in a closed tube and stored at room temperature for 2 h ± 15 min prior to analysis. Then they were introduced in tubes containing 9 mL of half Fraser broth (BIOKAR Diagnostics, BM01608). Tubes were mixed using vortex for 10 s before and incubated at 30 ± 1°C for 24 to 26 h. After the incubation period, a 0.1 mL aliquot of the primary enrichment was transferred in 10 mL of Fraser broth (BIOKAR Diagnostics, BM01308). The Fraser broth was incubated at 37 ± 1°C for 24 ± 2 h.

  From the primary enrichment, a loopful was streaked onto two selective agars: PALCAM agar plates (BIOKAR Diagnostics, BK145HA; BS00408) and chromogenic COMPASS^®  Listeria agar (BIOKAR Diagnostics, BM12408). In the same way, the second enrichment performed in Fraser broth was streaked onto the same selective agars. Plates were incubated at 37 ± 1°C for 24 ± 2 h and then 24 ± 2 h of overtime.

  For confirmation, up to five typical colonies for each sample were selected and streaked onto a nonselective agar plate (Tryptone Soya Yeast Extract Agar or TSYEA, BIOKAR Diagnostics, BK224HA). Plates were incubated at 37 ± 1°C for 18–24 h.

  Three confirmation tests were performed for each presumptive sample on isolated colonies: Beta-hemolysis test by streaking an isolated colony on tryptic soy agar plates containing 5% vol/vol of sterile sheep blood. Plates were incubated at 37 ± 1°C for 24 ± 2 h and plates were examined for hemolysis reaction. L-rhamnose and D-xylose tests were performed using carbohydrate utilization broths. One milliliter of each broth containing the specific sugars was inoculated with a loopful of culture. Cultures were incubated at 37 ± 1°C for 48–72 h. The change of color from purple to yellow was noted.

  For the N-Light L. monocytogenes assay, phosphate-buffered saline (pH 7.2 ± 0.2) premoistened swabs (FLOQSwab—COPAN) were used. Environmental surfaces were swabbed using firm and even pressure vertically (approximately 10 times), and then the sampler flipped and the other side used to sample horizontally (approximately 10 times) and diagonally (approximately 10 times). Swabs were introduced in a closed tube and stored at room temperature for 2 h ± 15 min prior to analysis. Then they were transferred into specific tubes containing 2 mL of NEMIS enrichment broth (NEB) and vigorously shaken by hand for 10 s. The swab was removed and discarded. Tubes were incubated at 37 ± 1°C for 24 h using a heating block. After incubation, an AquaSpark tablet was introduced in each enrichment tube and dissolved for 30 s by vortexing. Tubes were incubated for 10 min at room temperature (23 ± 2°C) before luminescence read out (Vitl; Lu mini).

  For presumptive positive samples, a loopful was streaked onto the two selective agar plates used for the ISO reference method (PALCAM agar plates and COMPASS Listeria agar). Plates were incubated at 37 ± 1°C for 24 ± 2 h and then 24 ± 2 h of overtime. Then confirmation was carried out as described for the ISO reference method.

• Results.—For each type of surface, the probability of detection (POD), defined as the number of positive outcomes divided by the total number of trials, was calculated for the N-Light L. monocytogenes assay (presumptive results and confirmatory results) and the ISO reference method (6). The desired fractional data sets for the low-level inoculated test portions were obtained for the three tested matrixes. For all matrixes, all uninoculated control test portions produced negative results by both methods. For all matrixes and all levels of inoculation, all presumptive positive N-Light L. monocytogenes assay results were confirmed; there were no false-positive results in the study (Table 4). All matrixes passed POD statistical analysis. Concerning the comparison between N-Light L. monocytogenes assay and the ISO reference method, for each matrix and each level of inoculation, no significant difference in the number of positive results was observed between the two methods (Table 5).

Results and Discussion

The N-Light L. monocytogenes assay detected all strains of L. monocytogenes tested during the inclusivity study. These tested strains belong to 12 different serotypes. The N-Light L. monocytogenes assay did not detect 30 strains of non-L. monocytogenes in which other species of Listeria were tested. The specificity of the assay was therefore validated according to the inclusivity/exclusivity study.

On stainless steel without competitive flora, the N-Light L. monocytogenes assay detected significantly more positive sample than the reference method for the low inoculation. When stainless steel was co-inoculated with L. monocytogenes and E. faecalis, there was no significant difference between N-Light L. monocytogenes assay and the ISO reference method. In the same way, there was no significant difference between the two methods when plastic or ceramic were used as environmental surfaces.

Conclusions

The data from this study supports the product claim that the NEMIS Technologies N-Light L. monocytogenes assay can detect L. monocytogenes from environmental surfaces when using the Lu-mini luminometer. The results obtained by the POD analysis of the method comparison study demonstrated that there were no statistically significant differences between the number of positive samples detected by the N-Light L. monocytogenes assay and the ISO reference method from environmental surfaces.

Acknowledgments

We would like to thank Nadine Heinrich, Giverny Ganz, and Anne Flore Imhaus for their contribution to the development of this assay.

Submitting Company

NEMIS Technologies

Überlandstrasse 109

8600 Dübendorf, Switzerland

Independent Laboratory

Q Laboratories

1930 Radcliff Drive

Cincinnati, OH, 45204

Reviewers

Yi Chen

US Food and Drug Administration

Center for Food Safety and Applied Nutrition

College Park, Maryland MD, 20740

Catharine Carlin

Mérieux NutriScience

Michiagan Ave.

Chicago, IL 60605

Salvatore Parisi

Al Balqa’ Applied University

Al-Salt 19117, Jordan

Sicily, Italy

Conflict of Interest

None declared.