Laporan Validasi Listeria-AOAC

RapidChek for the Detection of Listeria species in a Variety of Foods and
Select Environmental Surfaces

Abstract
Two RapidChek methods for the enrichment and detection of Listeria spp. in a variety of foods
and selected environmental surfaces have been validated as referenced to standard USDA/FSIS
and FDA/BAM cultural methods. The RapidChek Listeria lateral flow method (referred as LFD
method) uses a proprietary RapidChek Listeria enrichment media for a one-step 40hr enrichment
at 30oC, and detects Listeria on a lateral flow device in 10 min. The Rapidchek Listeria cultural
method also uses the same proprietary media as a one-step enrichment and detects Listeria by
streaking on selective plates as recommended by the standard cultural methods. The food
matrices tested included deli turkey, pepperoni, roast beef and hot dog, in which the USDA/FSIS
cultural method for Listeria was used as reference. The foods also included smoked fish, cooked
shrimp, milk, ice cream, potato salad and soft cheese, in which the FDA/BAM cultural method
for Listeria was used as a reference method. Three types of surfaces: stainless steel (1x1 in.),
painted concrete (1x1in.) and rubber (4x4in.) were tested using the reference of USDA/FSIS
method. A total of 10 Listeria strains/serotypes from 3 species were used to spike foods and the
surfaces as specified. Cells from each culture were spiked to one type of food at a target level of
1-10 cfu per 25g sample. Environmental samples were spiked at a level of 5.5 x 102 – 1 x 104
per surface (1sq inch for swabs, 4 sq. inches for sponge). A total of 260 spiked samples per
method (N=(10 foods + 3 surfaces) x 20 replicates x 1 method = 260) were tested, resulting in
189, 191 and 169 positives reported with the RapidChek LFD method, RapidChek cultural
method and reference methods, respectively. Non-spiked samples from all food and
environmental samples were reported as negative for Listeria spp. by all methods. The overall
method agreement of the RapidChek LFD method to the reference methods was 90%, while the
agreement between the RapidChek cultural method and reference methods was 89%. In
addition, the RapidChek LFD assay was evaluated with 50 Listeria strains and 30 non-Listeria
bacteria strains common to these food types. The results showed that the assay detected all the
Listeria strains and none of the non-Listeria strains, indicating a 100% sensitivity (inclusivity)
and a 100% specificity (exclusivity). These data demonstrated that RapidChek Listeria LFD
method, as well as the RapidChek Listeria cultural method perform as well as the USDA/FSIS
and FDA/BAM method for the detection of Listeria species in a variety of foods and select
environmental surfaces.
Method Authors
Jingkun Li, Carolyn Figard, Meredith Sutzko, Ly Tran and George Teaney
Strategic Diagnostics Inc., 128 Sandy Drive, Newark, Delaware 19713-1147
Submitting Company
Strategic Diagnostics Inc., 111 Pencader Drive, Newark, Delaware 19702
Independent Laboratories
Silliker Corporate Research Center, 160 Amory Drive, South Holland, IL 60473
1
1.0 Scope of Method
1.1 Target organisms – Listeria species
1.2 Matrices – Deli turkey, pepperoni, roast beef, hotdogs, smoked fish, cooked
shrimp, pasteurized whole milk, ice cream, potato salad, soft cheese, stainless
steel, painted concrete, and rubber.
1.3 Summary of validated performance claims – The Lateral Flow Device (LFD) test
Method for Listeria species was evaluated and was shown to have 100%
specificity (exclusivity) and 100% sensitivity (inclusivity) in this study. The
accuracy of the LFD test used with the 40hr proprietary enrichment system
relative to the reference methods averaged 137%. The method agreement
between the LFD test method and the USDA/FSIS and FDA/BAM methods for
the detection of Listeria in a variety of foods and select environmental surfaces
averaged 90% with the test method reporting 189 positive samples and the
reference methods reporting 169 positive samples. The RapidChek Listeria
media has also been evaluated for use with the selective agar isolation procedure
outlined in FDA Bacteriological Analytical Manual, 8th Edition, January 2003,
Chapter 10, and in USDA/FSIS Microbiology Laboratory Guidebook , 3rd Edition,
Rev #1, 9/6/99. Using the MOX agar detection system outlined in these
procedures the RapidChek cultural method accuracy averaged 138%, with an
average method agreement of 89%.
2.0 Definitions
2.1 Sensitivity – Rate of sensitivity = (No. of LFD Test Positives)/(No. Confirmed

Positives) × 100
2.2 Specificity – Rate of specificity = (No. of LFD Test Negative)/(No. Confirmed

Negatives) × 100
2.3 Method Agreement. – Method Agreement = [1- (No. of Correct LFD Test – No.
of Correct Reference Method / Total No. Method Samples)] × 100.
2.4 Accuracy – (No. of Confirmed Test Positives/Total No. Reference Positives) ×

100
3.0 Principle
3.1 The LFD Test for Listeria species can be used in combination with the proprietary
RapidChek Listeria enrichment system for a rapid 40 hour test. After enrichment, an
aliquot of sample broth is dispensed into a 12mm x 75mm test tube and boiled for 5
minutes. Once the sample has cooled, a test strip is added directly to the tube. The
sample flows up the strip through a zone containing antibody coated colloidal gold
reagents specific to Listeria species. If antigens are present in the sample, they will
bind to the antibody-conjugates to form an antigen/antibody complex. As this
complex migrates through the nitrocellulose matrix, it passes a zone of anti-Listeria
antibody. If antigen is present, the complex is captured in this zone and is visualized
2
by the formation of a red line. A second zone on the membrane is designed to capture
any antibody-gold complex not bound in the first zone. As a result, when Listeria
antigen is present, the formation of 2 red lines is observed, whereas when no Listeria
is present, only 1 line forms.
4.0 General Information
4.1 Listeria species are ubiquitous in nature found in environments such as plants, soil,
animals, water, dust and silage. Since Listeria species are frequently found in
slaughter animals, and thus in raw meat and poultry, the organism can be
continuously introduced into the processing environment (1, 2). The ability of this
organism to cross contaminate food contact surfaces, equipment, floors, drains etc (3)
and to grow under adverse condition including extremes of heat, salt and temperature
has been well documented. The organism has also been found to thrive in damp
environments potentially forming biofilms in the processing environment making it
difficult to eliminate during cleaning and sanitizing processes.
Among the six species of Listeria commonly found in foods, L. monocytogenes and L.
ivanovii are the pathogenic species, capable of causing human illness, with the L.
monocytogenes being most commonly linked to foodborne outbreaks (4). The
symptoms of listeriosis vary, ranging from mild flu like conditions to more severe
infections of the blood and brain such as septicemia and meningitis, encephalitis).
Although the infectious dose remains unclear, host susceptibility plays a major role in
illness with immunocompromised individuals being the most susceptible populations
(5).
In order to prevent occurrence of listeriosis, government regulations stipulate the total

absence of Listeria species in food, as presence of the organism is indicative of
insufficient hygiene practices in the handling of raw materials and finished products.
Routine testing of high risk products and other ready to eat foods that may support the
growth of Listeria is therefore important , for both the prevention of illness and to
avoid costly recalls within the food industry. Conventional enrichment and isolation
procedures for Listeria in foods tend to be slow and laborious, requiring 3-5 days for
presumptive identification (6, 7). Therefore, a detection method with similar or better
performance, but which reduces the amount of labor and cost associated with these
methods, would greatly benefit the control of Listeria in the food industry.
5.0 Test Kit Information
5.1 Kit Name – RapidChek Listeria test kit, includes 50 lateral flow devices,
disposable pipettes, and test tubes to be used with the RapidChek Media for
Listeria enrichment media.
5.2 Catalog Number – 7000171
5.3 Ordering Information – Strategic Diagnostics Inc., 111 Pencader Drive, Newark,
DE 19702; Phone: (800) 544-8881 or www.sdix.com
3
6.0 Media and Reagents
6.1 RapidChek Listeria Lateral Flow assay
6.2 Enrichment
6.2.1 RapidChek Media and Supplement for Listeria (7000172 & 7000173)–
(Strategic Diagnostics Incorporated, Newark, DE). Dissolve 53g of the
base media in one liter of sterile distilled or deionized water pre-warmed
to 30oC. Add 1.0g/L of the supplement powder supplied. RapidChek
Media is not sterile and therefore should be used within 6 hours for best
results. Alternatively, rehydrate the base media as described above and
autoclave at 121 oC for 15 min. Allow base media to cool and add
supplement powder at 1.0g/L.
6.2.2 Buffered Listeria Enrichment Broth Base (BLEB) - (Oxoid).

monopotassium phosphate, 1.35g/L
dipotassium phosphate, 9.6g/L
trypticase peptone, 17g/L
phytone peptone, 3g/L
glucose, 2.5g/L
sodium chloride, 5g/L
disodium phosphate, 2.5g/L
yeast extract, 6.0g/L
sodium pyruvate, 110mg/L
Dissolve 47g of powder media in one liter of sterile distilled or deionized

water. Autoclave for 15 minutes at 121oC. Cool to room temperature.
Add sodium pyruvate as a 10% (w/v) aqueous filter sterilized solution
(11.1mL/L).
6.2.3 BLEB Supplement (selective agents):

nalidixic acid, sodium salt (ICN), 40mg/L
acriflavin HCl (ICN), 10mg/mL
cycloheximide (ICN), 50mg/mL
Prepare nalidixic acid and acriflavin HCl as 0.5% (w/v) stocks in distilled
water. Prepare cycloheximide supplement as a 1.0% (w/v) stock in 40%
(v/v) ethanol in water. Add 0.455 ml nalidixic acid, 1.8 mL acriflavin
HCl, and 1.15 mL cycloheximide filter sterilized stock solutions to each
sample as described in section 10.1.4.
4
6.2.4 Modified University of Vermont Broth (UVM) – (Remel)
casien peptone, 5.0g/L
beef extract, 5.0g/L
sodium chloride, 20.0g/L
meat peptone, 5.0g/L
esculine, 1g/L
acriflavine HCl, 0.012g/L
nalidixic acid, 0.02g/L
add 47g of powder media to 1L of deionized water. Autoclave for 15

minutes at 121oC. Cool to room temperature.
6.2.5 Fraser Broth – (Difco Becton Dickinson)
casien peptone, 5.0g/L

proteose peptone, 5.0g/L
esculine, 1g/L
acriflavine HCl, 0.024g/L
nalidixic acid, 0.02g/L
lithium chloride, 3.0g/L
add 55g powder media to 1 L of deionized water and dissolve media and
dispense 10 ml into sterile tubes, autoclave 116°C for 15 mins. Add
0.5g/L Ferric Ammonium Citrate.
6.2.6 Dey-Engley neutralizing broth (D/E) – (Difco)

pancreatic digest of casein, 5g/L
dextrose, 10.0g/L
sodium thioglycollate, 1.0g/L
sodium thoisulfate, 6.0g/L
sodium bisulfite, 2.5g/L
polysorbate 80, 5.0g/L
lecithin, 7.0g/L
bromocresol purple, 0.02g/L
add 39g of powder media to 1L of deionized water. Autoclave for 15

minutes at 121oC. Cool to room temperature.
5
6.2.7 MOX Agar – (Difco Becton Dickinson)
Columbia Blood Agar Base, 39.0g/L
agar, 2.0g/L
esculin, 1.0g/L
ferric ammonium citrate, 0.5g/L
lithium chloride, 15.0g/L
Supplements: (Difco Becton Dickinson)

colistin methane sulfonate, 0.01g/L
moxalactam, 0.02g/L
add 57.5g of powder media to 1L of deionized water. Autoclave for 15 minutes at

121oC. Cool to 45-50C. Add 10mL of filter sterilized supplement solution. Mix
and dispense.
6.3 Disposable 200 µl pipette tips
6.4 Filtered stomacher bags
6.5 10uL and 1uL sterile, disposable loops (FisherBrand)
6.6 Vortex mixer
6.7 Trypticase Soy Agar – Yeast Extract (TSAYE) (Difco, 236950, 212750)
pancreatic digest of casein, 15.0g/L
enzymatic digest of soybean meal, 5.0g/L
agar, 15.0g/L
add 57.5g of powder media to 1L of deionized water. Autoclave for 15 minutes at

121oC. Cool to 45-50C. Mix and dispense.
6
6.8 Horse Bilayer Columbia Agar – (Remel, 01477)
bottom layer:
casein peptone, 12.0g/L
corn starch, 1.0g/L
agar, 13.5g/L
top layer:
casein peptone, 12.0g/L
corn starch, 1.0g/L
horse blood, 50mL/L
agar, 13.5g/L
6.9 Motility Test Media (MTM)
beef extract 3g/L

peptone, 10g/L
NaCl, 5g/L
agar, 4g/L
Heat to dissolve agar, dispense 8mL to glass tubes, cap and autoclave for 15 min
at 121C.
6.10 API Listeria ID Kit – (bioMerieux)
6.11 Hydrogen peroxide solution, 3%
6.12 Gram stain reagents (Fisher Scientific)
6.13 Immersion Oil
6.14 Microscope slides, glass
6.15 Cover slips, glass
6.16 Non-bacteriocidal 7.5x4x0.3cm sterile cellulose sampling sponge pre-moisten

with 10mL D/E neutralizing broth – (Solar Biologicals, BS-10DE)
6.17 Sterile cotton-tipped swab – (Fisher)
7.0 Apparatus
7
7.1 RapidChek Listeria Lateral Flow devices( Figure 1).
7.2 Micropipet – Capable of accurately dispensing 400 µl.
7.3 Stationary incubator – Capable of holding temperature at 30oC+/- 0.5 for 40-48
hours.
7.4 Stationary incubator – Capable of holding temperature at 35oC +/-1.0 for 20-24
hours.
7.5 Stomacher – Seward 400 Circulator (Seward, London, England) - for thorough
mixing of food samples in enrichment broth.
7.6 Top loading balance – To weigh 25 g ( + 0.1g) test samples.
7.7 Zeiss phase contrast microscope.
8.0 Standard Reference Material
8.1 Chapter 4 entitled Isolation and identification of Listeria from meat, poultry and
egg products (revision #1; 1/10/01))” in USDA/FSIS Microbiology Laboratory
Guidebook , 3rd Edition, Rev #1, 9/6/99. Web page:
http://www.fsis.usda.gov/ophs/microlab/mlgbook.html
8.2 Chapter 10 entitled “Detection and Enumeration of Listeria monocytogenes in

Foods” in FDA Bacteriological Analytical Manual, 8th Edition, January 2003.
Web page: http://www.cfsan.fda.gov/~ebam/bam-10.html
9.0 Safety Precautions
9.1 Live bacteria may be infectious and pose a significant health hazard. All bacterial
cultures should be handled with caution and biohazard waste should be disposed
of appropriately.
10.0 Sample Preparation
10.1 Food Samples
10.1.1 Aseptically weigh a representative 25 g sample and add to filtered

stomacher bag.
10.1.2 Add 225 ml of either BLEB (FDA reference method), UVM (USDA
reference method) or RapidChek Media (Test method) pre-warmed to 30
o
C.
10.1.3 Stomach samples for approximately 1-2 minute.
8
10.1.4 Incubate FDA reference method (BLEB) samples at 30oC for 4 h then add
selective agents and continue incubation at 30oC for a total time of 48 h.
10.1.5 Incubate USDA (UVM) samples at 30+/-2oC for 22 +/-2 h in a stationary

incubator. Transfer 0.1+/- 0.02mL of the UVM enrichment to 10+/-
0.5mL FB (with supplements) and incubate at 35+/-2oC for 26 +/- 2 h.
10.1.6 Incubate Proprietary Media samples at 30oC for 40 h in a stationary

incubator.
10.2 Isolation and Confirmation Procedure
10.2.1 RapidChek Methods: After 40h incubation, test sample using RapidChek
test strip as described below. Streak all samples to MOX agar plates and
incubate for 24-48 h at 35+/-2oC.
10.2.2 BAM Reference Method: After 24 and 48 h streak BLEB enrichment to

MOX agar plates and incubate for 24-48 h at 35+/-2oC.
10.2.3 FSIS Reference Method: After 24 and 48 h streak UVM and FB

enrichments to MOX agar plates and incubate for 48h at 35 +/-2oC.
10.2.4 Samples containing Listeria like MOX colonies are confirmed according
to the FDA/BAM or FSIS/USDA procedure. In brief, the FDA
confirmation involved transferring well isolated, typical Listeria like
colonies to TSAYE and confirming for beta-hemolysis, motility using
MTM, catalase and gram stain testing in addition to biochemical tests in
the form of API Listeria testing. Similarly, the USDA confirmation
methods employed included a determination of tumbling motility, beta-
hemolysis and biochemical confirmation using API Listeria testing.
11.0 Set Up and Operation
11.1 Label each tube with the appropriate sample identification.
11.2 Take one pipette from the bag. Squeeze and hold the bubble on the top of the
pipette and place in the sample enrichment.
11.3 Release the bulb and the required volume of solution (400uL) will rise in the
barrel of the pipette.
11.4 Hold the pipette over the sample reservoir on the cassette and add the entire
sample volume of the pipette barrel by squeezing the bulb. Discard the used
pipette.
11.5 Heat tube to 100 oC for 5 min and allow sample to cool.
11.6 Insert test strip and let develop for 10 minutes.

9
12.0 Interpretation and Test Result Report
12.1 After a 10 minute time period, the appearance of one red line (control line) on the
LFD Test indicates a negative result. The appearance of two red lines ( the
control line and the test line) on the LFD Test indicates a positive result (Figure
1).
13.0 Independent Validation Studies
13.1 Independent validation studies were conducted by Silliker Laboratories in South

Holland, Illinois, under the direction of the AOAC Research Institute.
13.2 Method Comparison Study – Roast beef, rubber surfaces, and ricotta cheese were
analyzed by the LFD method as well as the RapidChek cultural method to
determine the agreement of the kit performance relative to the reference method
for these matrices. Replicate samples at two inoculum levels (zero and low) were
examined for each matrix. Refer to Tables 4 and 5 for a summary of the
independent validation study results.
13.2.1 Methodology
13.2.1.1 Roast Beef
L. innocua (ATCC 33090) was grown in 10 ml TSB

broth at 35°C for 18 h. Two thousand grams of roast
beef was inoculated with a heat stressed (50 oC for 10
min) low inoculum (1-10 cfu/25g) based on microbial
plate count results and homogenized by mixing
manually in a large stomacher bag for 5 minutes.
Samples were held at 4oC for 48 h. A 3 tube 4 level
MPN (most probable number) was set up using the
USDA enrichment procedure (described above), to
determine cell levels in the sample after the stress
period. In short 100, 10, 1, and 0.1g of inoculated roast
beef were stomached in UVM media, incubated at
30+/- 2°C for 22h , transferred to FB and incubated an
additional 26 h at 35 +/- 2°C. Samples were streaked to
MOX agar plates and confirmed as described above.
The 100, 10 and 1g samples were subsequently used to
determine the MPN.
Inoculated meat was divided into two sets of twenty 25

g samples. One set was stomached with 225 ml of the
RapidChek Test Media for 1 minute and incubated for
40 h at 30+/- 2°C. Samples were heat treated and tested
on the LFD as previously described.
A second set of twenty 25 g samples of inoculated

turkey were enriched in 225 ml of UVM, which was
10
incubated at 30°C +/- 2°C for 22 h. Samples were then
transferred to FB for an additional 26h incubation at
35+/- 2°C according to the USDA/FSIS reference
protocol for Listeria monocytogenes, Chapter 8 (7).
All samples were confirmed using the standard cultural

and biochemical tests recommended by USDA/FSIS as
described in section 10.1.9
13.2.1.2 Ricotta Cheese
L. monocytogenes 4ab (SDI -18c) was grown in 10 ml

TSB broth at 35°C for 18 h. Two thousand grams of
Ricotta Cheese was inoculated with a heat stressed (50
o
C for 10 min) low inoculum (1-10 cfu/25g) based on
microbial plate count results and homogenized by
mixing manually in a large stomacher bag for 5
minutes. Samples were held at 4oC for 48 h. A 3 tube
4 level MPN (most probable number) was set up using
the FDA enrichment procedure (described above), to
period. In short 100, 10, 1, and 0.1g of inoculated
cheese were stomached in BLEB base media, incubated
at 30+/- 2°C for 4h , followed by an addition of
antibiotic supplements and incubated an additional 44 h
at 30 +/- 2°C. Samples were streaked to MOX agar
plates and confirmed as described above. The 100, 10
and 1g samples were subsequently used to determine
the MPN.
Inoculated cheese was divided into two sets of twenty

25 g samples. One set was stomached with 225 ml of
the RapidChek Test Media for 1 minute and incubated
for 40 h at 30+/- 2°C. Samples were heat treated and
tested on the LFD as previously described.

cheese were enriched in 225 ml of BLEB, which was
supplemented with an addition of selective agents and
incubated an additional 44 h at 30 +/- 2°C as described
in the FDA reference protocol for Listeria
monocytogenes, Chapter 10 (6).

and biochemical tests recommended by FDA/BAM as
11
13.2.1.3 Rubber Environmental Surfaces
L. monocytogenes 4ab (SDI -18) was grown in 10 ml

TSB broth at 35°C for 18 h. Four square inch rubber
surfaces were inoculated based on microbial plate count
with 1000 cfu/4 inch2 in 10% skim milk and allowed to
dry for 24 hours.
Sponge samples were taken using sponges pre-

moistened with D/E broth. The entire 4 inch2 surface
was scrubbed with a single sponge for approximately
30 seconds. One set of 25 sponges (20 from inoculated
surfaces, 5 from non-inoculated surfaces) was
stomached with 100 ml of the RapidChek Test Media
for 1 minute and incubated for 40 h at 30+/- 2°C.
Samples were heat treated and tested on the LFD as
previously described.
A second set of 25 sponge samples (20 from

inoculated surfaces, 5 from non-inoculated surfaces)
was enriched in 225 ml of UVM, which was incubated
at 30°C +/- 2°C for 22 h. Samples were then transferred
to FB for an additional 26h incubation at 35+/- 2°C
according to the USDA/FSIS reference protocol for
Listeria monocytogenes, Chapter 8 (7).

13.2.2 Results:
13.2.2.1 In the External laboratory study all of the non-

inoculated roast beef samples reported negative results
(Table 4). Fourteen (14) of 20 inoculated roast beef
samples were reported and confirmed positive using the
Test LFD and cultural methods and 9 of 20 samples
were reported and confirmed positive by the USDA
reference method. No false positive or false negative
results were reported by either of the RapidChek
methods. No significant differences were observed
between test and reference methods (p<0.05).
13.2.2.2 RapidChek methods reported 12 of the 20

inoculated ricotta cheese samples as positive where as
the FDA reference method reported 13 of the 20
samples as positive. No false negative or false positive
samples were reported by either RapidChek method.
All non-inoculated samples were reported and
12
confirmed negative. No significant differences were
observed between test and reference methods (p<0.05).
13.2.2.3 In the rubber environmental surface matrix the

RapidChek methods reported 9 of the 20 samples as
positive whereas the reference method reported and
confirmed 2 of the 20 samples as positive (Table 5).
All non-inoculated samples were reported and
confirmed to be negative. The test methods were both
found to be statistically different from the reference
method (p<0.05)
14.0 Internal Validation Studies
14.1 Inclusivity Study

14.1.1 Internal validation studies were conducted by Strategic Diagnostics Inc.
128 Sandy Dr. Newark, DE 19713.
14.1.1 Fifty (50) Listeria spp. strains (Table 1) were analyzed to determine the
sensitivity of the LFD Test.
14.1.2 Methodology
14.1.2.1 Fifty (50) Listeria spp. strains were inoculated into

individual 10 ml aliquots of the RapidChek Listeria
broth and incubated at 30+/-1.0 oC for 24 h. After
enrichment, samples were tested on the LFD Test as
described previously.
14.1.3 Results
14.1.3.1 Results from the Inclusivity study are summarized in Table 3.

As can be seen, the LFD Test was found to have a sensitivity of
100% for detecting specific strains of Listeria from a variety of
sources including clinical, environmental and food isolates.
14.2 Exclusivity Study
14.2.1 Thirty (30) non-Listeria strains (Table 3) were tested to determine the
specificity of the kit.
14.2.2 Methodology
14.2.2.1 Strains were grown at 35+/-1.0 oC for 24 h in 10 ml of TSB

broth. After incubation, samples were evaluated using the LFD
Test as described previously. Any samples testing positive
were re-grown in RapidChek Listeria broth incubated at 30+/-
1.0 oC for 24 h, and tested on the LFD as described previously.
13
14.2.3 Results
14.2.3.1 Exclusivity results are summarized in Table 3. Of the 30 non-

Listeria strains tested, 1 isolate, Staphylococcus aureus,
reported a positive result when grown in BHI. However, the
same strain tested negative when grown in RapidChek resulting
in a method specificity of 100%.
14.3 Method Comparison Study
14.3.1 Deli turkey, hotdogs, pepperoni, smoked fish, cooked shrimp, whole milk,
ice cream, potato salad, painted concrete and stainless steel were analyzed
by the LFD method as well as the RapidChek cultural method to
determine the agreement of the kit performance relative to the
corresponding reference methods for each food and surface matrix
examined. Replicate samples at two inoculum levels (zero and low) were
examined for each matrix.
14.3.2 Methodology
14.3.2.1 Deli Turkey
L. monocytogenes 1/2a (ATCC # 51774) ) was grown

in 10 ml BHI broth at 35°C for 18-24 h. Two thousand
grams of deli turkey was inoculated with a heat stressed
(50 oC for 10 min) low inoculum (1-10 cfu/25g) based
on microbial plate count results and homogenized by
shaking and mixing manually in a large stomacher bag
for 5 minutes. Samples were held at 4oC for 48 h. A 3
tube 4 level MPN (most probable number) was set up
using the USDA enrichment procedure (described
above), to determine cell levels in the sample after the
stress period. In short 100, 10, 1, and 0.1g of
inoculated deli turkey were stomached in UVM media,
incubated at 30+/- 2°C for 22h , transferred to FB and
incubated an additional 26 h at 35 +/- 2°C. Samples
were streaked to MOX agar plates and confirmed as
described above. The 100, 10 and 1g samples were
subsequently used to determine the MPN.
Inoculated meat was divided into two sets of twenty 25


turkey were enriched in 225 ml of UVM, which was
14
transferred to FB for an additional 26h incubation at
35+/- 2°C according to the USDA/FSIS reference
protocol for Listeria monocytogenes, Chapter 8 (7).

14.3.2.2 Hotdogs
Listeria welshimeri (ATCC # 35897) was grown in 10

ml BHI broth for at 35°C for 18-24 h. All inoculation
and enrichment procedures were carried out as
described in Section 14.3.2.1.
14.3.2.3 Pepperoni
L. monocytogenes 3b (SDI 15) was grown in 10 ml BHI

broth for at 35°C for 18-24 h. All inoculation and
enrichment procedures were carried out as described in
Section 14.3.2.1.
14.3.2.4 Smoked Fish
L. monocytogenes 7 (SDI 23) was grown in 10 ml BHI

broth at 35°C for 18-24 h. Two thousand grams of
smoked fish was inoculated with a heat stressed (50 oC
for 10 min) low inoculum (1-10 cfu/25g) based on
microbial plate count results and homogenized by
mixing manually in a large stomacher bag for 5
minutes. Samples were held at 4oC for 48 h. A 3 tube
4 level MPN (most probable number) was set up using
the FDA enrichment procedure (described above), to
period. In short 100, 10, 1, and 0.1g of inoculated fish
were stomached in BLEB base media, incubated at
30+/- 2°C for 4h , followed by an addition of antibiotic
supplements and incubated an additional 44 h at 30 +/-
2°C. Samples were streaked to MOX agar plates and
confirmed as described above. The 100, 10 and 1g
samples were subsequently used to determine the MPN.
Inoculated fish was divided into two sets of twenty 25

15
cheese were enriched in 225 ml of BLEB, which was
supplemented with an addition of selective agents and
incubated an additional 44 h at 30 +/- 2°C. as described
in the FDA reference protocol for Listeria

and biochemical tests recommended by FDA/BAM as
14.3.2.5 Cooked Shrimp
L. monocytogenes 7 (SDI 23) was grown in 10 ml BHI

broth at 35°C for 18-24 h. All inoculation and
Section 14.3.2.4.
14.3.2.6 Whole Milk
L. monocytogenes 4b (ATCC 19115) was grown in 10

ml BHI broth at 35°C for 18-24 h. All inoculation and
Section 14.3.2.4 with the following exception; 1 gallon
of pasteurized whole milk was inoculated as described
in 14.3.2.4.
14.3.2.7 Ice Cream
L. monocytogenes 3a (SDI 14) was grown in 10 ml BHI

Section 14.3.2.4 with the following exception;
inoculum was freeze stressed for 48h in the ice cream
matrix.
14.3.2.8 Potato Salad
L. monocytogenes 1/2b (SDI 12) was grown in 10 ml

BHI broth at 35°C for 18-24 h. All inoculation and
Section 14.3.2.4.
14.3.2.9 Painted Concrete
L. monocytogenes 4b (ATCC 19115) was grown in 10

ml BHI broth at 35°C for 18-24 h. One square inch
painted surfaces were inoculated based on microbial
16
plate count with 1x104 cfu/ inch2 in 10% skim milk and
allowed to dry for 24 hours.
Swab samples were taken using sterile swabs moisten

with D/E broth. The entire 1 inch2 surface was
scrubbed with a single swab for approximately 30
seconds. One set of 25 swabs (20 from inoculated
surfaces, 5 from non-inoculated surfaces) was added to
tubes containing 10 ml of the RapidChek Test Media
and incubated for 40 h at 30+/- 2°C. Samples were heat
treated and tested on the LFD as previously described.
A second set of 25 swab samples (20 from inoculated

surfaces, 5 from non-inoculated surfaces) were enriched
in 225 ml of of UVM, which was incubated at 30°C +/-
2°C for 22 h. Samples were then transferred to FB for
an additional 26h incubation at 35+/- 2°C according to
the USDA/FSIS reference protocol for Listeria

14.3.2.10 Stainless Steel
L. innocua (ATCC 33090) was grown in 10 ml BHI

Section 14.3.2.9 with the following exception; The
inoculum level applied as determined by direct plate
count was 5.5X102 cfu/ inch2. Samples were co-
inoculated with cultures of Bacillus subtillis at 4.4X102
cfu/ inch2, Enterococcus faecalis at 1.2X103 cfu/ inch2,
and Streptococcus oralis at 1.2X103 cfu/ inch2 as
determined by aerobic plate count.
14.3.3 Results
14.3.3.1 Results from the method comparison/repeatability study are

summarized in Tables 6 - 10. In Deli Turkey, the performance
of the LFD method with 40 h enrichment scheme was found to
comparable to the RapidChek cultural method of detection with
the LFD test detecting 14 of the 20 inoculated samples as
positive and the cultural method reporting 15 of the 20
inoculated samples as positive. However one of the samples
determined to be negative by the LFD method was confirmed
to be positive on MOX selective agar. The reference method
reported positive results from 13 of the 20 inoculated samples.
17
Non-inoculated samples were found to be negative by both the
RapidChek methods as well as the USDA/FSIS method. The
LFD and cultural methods reported 108% and 115% accuracy
respectively. No statistical differences (p<0.05) between test
and reference methods were reported.
14.3.3.2 A similar scenario was evident for hotdog samples, with all
non-inoculated samples returning negative results by the
RapidChek methods and USDA/FSIS method. The LFD and
cultural method recovered and positively identified 16 of the
20 inoculated samples. The reference method also recovered 16
of the 20 inoculated samples. 100% accuracy and method
agreement was reported for both the methods tested for this
matrix. No statistical differences (p<0.05) between test and
reference methods were reported.
14.3.3.3 For Pepperoni, both the RapidChek methods recovered 10 of

the 20 inoculated samples, all of which were confirmed
culturally to be L. monocytogenes. The USDA/FSIS method
positively identified 12 of the 20 samples inoculated with
Listeria. All of the non-inoculated samples returned negative
results for all methods. For this matrix, a 83% accuracy and
90% method agreement was reported with both detection
methods examined. No statistical differences (p<0.05)
between test and reference methods were reported.
14.3.3.4 In the Smoked Fish study all of the non-inoculated samples

were reported negative. The LFD and cultural methods
positively identified and confirmed 14 of the 20 inoculated
samples. The FDA reference method reported 16 of the 20
inoculated samples as positive resulting in 90% method
agreement and 87% accuracy. No statistical differences
(p<0.05) between test and reference methods were reported.
14.3.3.5 In the cooked shrimp study the RapidChek methods reported

and confirmed 11 of the 20 inoculated samples, to be positive
as did the reference method. All non-inoculated samples were
reported as negative by all methods. No statistical differences
14.3.3.6 Results from potato salad reported the RapidChek LFD method
recovering 16 of the 20 spiked sampled while the RapidChek
cultural method reported 17 of the 20 spiked samples as
positive. One of the samples determined to be negative by the
LFD method was confirmed to be positive on MOX selective
agar. The reference method reported 18 of the 20 samples as
positive. These data represent 89 and 94% accuracy and 90%
and 95% method agreement for the LFD and cultural methods
respectively. None of the non-inoculated samples were
reported as positive by any of the methods. No statistical
18
differences (p<0.05) between test and reference methods were
reported.
14.3.3.7 RapidChek LFD and cultural methods reported and confirmed

17 of the 20 inoculated whole milk samples as positive where
as the FDA/BAM reference method reported 18 of the 20
samples as positive resulting in 94% accuracy and 95% method
agreement for the test methods. All non-inoculated samples
were reported as negative by all methods. No statistical
differences (p<0.05) between test and reference methods were
reported.
14.3.3.8 In ice cream, RapidChek methods reported and confirmed 20

of the 20 inoculated samples as positive and the Reference
method reported 16 of the 20 samples as positive. 125%
accuracy and 80% method agreement were reported for the
both RapidChek methods. All non-inoculated samples were
reported as negative by all methods. No statistical differences
14.3.3.9 Swab sample results from painted concrete reported and

confirmed 18 of the 20 inoculated samples as positive for the
RapidChek methods and 10 of the 20 samples as positive using
the Reference method. These results equate to 180% accuracy
and 40% method agreement for both RapidChek methods.
Non-inoculated samples were reported to be negative by all
methods. The numbers of positives reported by the test
methods and the reference methods were statistically different
(p<0.05).
14.3.3.10 Lastly, the RapidChek methods recovered and confirmed 18 of

the 20 inoculated stainless steel surfaces and the reference
method reported 15 of the 20 samples as positive. Eighty five
(85) percent method agreement and 120% accuracy was
recorded for both RapidChek methods. All non-inoculated
samples were reported as negative by all methods. No
statistical differences (p<0.05) between test and reference
methods were reported.
14.4 Ruggedness Studies
14.4.1 The LFD Test is a very simple device to utilize. It is a compact diagnostic
strip which when inserted into an aliquot of the enriched sample, runs
unattended in 10 minutes. Therefore, ruggedness testing for this study
involved variation in temperature at which the device was tested,
variations in times at which the strip was read, variations in sample
preparation times. Ruggedness testing additionally involved variations in
sample volume added to the LFD Test, variability in time lots of the test
19
strip used, and stability of test line after expiration of reading time has
occurred.
14.4.2 Methodology
14.4.2.1 Temperature and Device Read Time Latitude Study - LFD

Tests were removed from their desiccant can and placed at
4°C, 25°C and 45°C to mimic potential temperature ranges at
which the devices could be tested by the user. Both inoculated
Listeria swab samples and non inoculated swab samples were
enriched with the 40 h enrichment and tested on the devices at
4°C, 25°C and 45°C. It is recommended in the package insert
information for the LFD Test that 10 minutes is required prior
to reading the strip and recording the result as negative for
Listeria. In addition to the assay run temperature evaluation,
this study was designed to determine what effect a variation on
reading the strip had on the end result of the assay. LFD Tests
from the temperature study described above were read at 5, 8,
10, 15, and 20 minute intervals.
14.4.2.2 Sample Volume Study - It is recommended in the package

insert for the LFD Test, that 400µl of the enriched sample be
added to the test tube prior to boiling. This study determined
the effect of variations in the sample volume used to run the
device. Ten (10) Listeria positive swab samples and 5 negative
swab samples enriched in the proprietary media were tested
with the device. Volumes of 300µl, 400µl, and 500µl, of
negative and positive samples were evaluated. LFD Tests were
run and evaluated at 10 minutes and observations made.
14.4.2.3 Lot to Lot Variability Study – Smoked Fish samples were

inoculated with L. monocytogenes 7 at one level (5.0
cfu/sample, 20 sample) and enriched in the proprietary media
for 40 h at 30°C. Five non-inoculated controls were also run
with this experiment. After incubation, samples were tested on
2 Production and 1 R&D lots of the LFD Tests to determine the
lot to lot variability of the strips.
14.4.2.4 Stability Study - The stability of the LFD Test was examined
over a period of 46 days. LFD Tests were stored at 4°C, 25°C,
37°C and 45°C under desiccated conditions. Devices from an
R&D Lot and 2 Manufacturing Lot were removed from their
respective storage temperatures and tested with cultures of L.
monocytogenes and L. innocua calibrators at appx. 1X106
cfu/ml . A negative control culture of Bacillus subtilis was also
examined. Samples were tested on the LFD Test at the
following time points, day 0, 7, 14, 20, 28, 35.
14.4.2.5 RapidChek Media Preparation Latitude – It is recommended in

the package insert that the end user prepare the RapidChek
20
media by dissolving the appropriate mass of powdered media
to the appropriate volume of autoclaved water. An alternative
to this preparation method is to prepare and autoclave the
media a day prior to the use. In this study, potato salad and
smoked fish were inoculates with 2 different serotypes of L.
monocytogenes, at levels ranging from 2.8 – 3.0 cfu/25g as
determined by plate count (Table 15). Samples were enriched
with RapidChek Listeria media prepared using both of the
methods described in the package insert. Twenty inoculated
samples were enriched from each food type, and were tested
using the RapidChek LFD, and confirmed as described above.
14.4.2.6 Sample Preparation Latitude- The Users Guide recommends

heat treating the sample at 100°C for 5 to 15 minutes in order
to sufficiently prepare the antigen in the sample to attain
maximum sensitivity. In this study, pure cultures of 14
Listeria cultures representing 14 different serotypes were
grown for 24h in RapidChek media at 30°C, cultures
enumerated using standard dilution plating techniques and
diluted to 1 X 10-6 in peptone water. Diluted samples were
subject to heat treatment at 100°C for 5, 10, 15 and 30 minutes
and subsequently run with the LFD’s.
14.4.2.7 Sponge enrichment volume latitude study - The users Guide

recommends enriching sponge samples in a 100mL volume of
RapidChek media. This study was designed to determine the
effect of using a significantly lower volume (60mL) of media
during the enrichment process. L. innocua (ATCC 33090) was
grown in 10 ml BHI broth at 35°C for 18-24 h. Three
inoculum levels were prepared and applied to the stainless steel
surface in 10% skim milk and allowed to dry for 24 hours. The
levels, as determined by direct plate count were 6.5X102 cfu/ 4
inch2, 2.2X102 cfu/ 4 inch2, and 1.3X102 cfu/ 4 inch2. Samples
were co-inoculated with cultures of Bacillus cereus at 4.2X103
cfu/ 4 inch2, Enterococcus durans at 6.9X104 cfu/ 4 inch2, and
Pseudomonas aerugin at 2.8X104 cfu/ 4 inch2 as determined by
aerobic plate count. Non-inoculated surfaces were prepared in
the same manner excluding L. innocua. Sponges wetted with
10mL of D/E broth were used to scrub each of the spiked
surfaces and placed into stomacher bags containing either 100
mL of 60 mL of RapidChek media. Samples were stomached
for approximately 1 minute and incubated for 40h at 30oC.
Following the enrichment, samples were tested as described in
section 11.
21
14.4.3 Results
14.4.3.1 The Listeria LFD test was found to function properly

throughout the temperature and timing ranges described in
section 14.4.2.1 (Table 11). Although in this study the correct
result was observed as early as 5 minutes, it remains the
recommendation of the manufacturer that a full ten minutes is
allowed in order to properly identify negative samples.
14.4.3.2 The minimum volume of sample required to properly run the

device is 300uL (Table 12). Sample volumes from 300 uL
through 500uL all returned the same assay result. Therefore
the manufacture recommends the use of the 400uL disposable
pipette supplied with the kit.
14.4.3.3 The Lot to Lot variability between two production lots and one
R&D lot was evaluated (Table 13). No significant Lot to Lot
differences were observed with the LFD product.
14.4.3.4 Results from the accelerated stability study report no decrease

in performance from either LFD Lot at any of the temperature
challenges over the course of the study (Table 14). These
results indicate that the LFD test will have 1 year stability at
room temperature and possibly longer duration under
refrigerated conditions.
14.4.3.5 The use of autoclaved RapidChek media does not appear to

effect the recovery of Listeria from the sample (Table 15).
Results from this study show 33 samples determined positive
using autoclaved media, and 35 samples determined positive
using non-autoclaved media.
14.4.3.6 Results from the sample preparation latitude study (Table 16)
indicate that a significant increase in assay sensitivity to
Listeria spp. is achieved with a sample boiling duration from 5
through 30 minutes.
14.4.3.7 There appears to be a good deal of latitude in the allowable

volume of media used for an environmental sponge enrichment
(Table 17). Data from this study indicate that there is minimal
difference between the use of 60mL and 100mL of enrichment
media.
22
15.0 Discussion
15.1 The RapidChek LFD and cultural methods have demonstrated excellent accuracy,
sensitivity and specificity throughout these studies. Overall method agreement
averaged 91% for the LFD and the cultural methods in food samples, though
when method agreement data were adjusted to reflect the cases where the
RapidChek methods recovered a greater number of positive samples than the
reference methods, 100% and 102% method agreement were reported,
respectively. In all 3 surface studies, the RapidChek methods reported greater
numbers of positives than the reference method with the corresponding method
agreement averaging 69% (131% when adjusted to reflect greater recovery).
Overall, there were no false positive results and 2 false negative results observed
using the LFD detection system (1% false negative rate, 0% false positive rate).
There were 2 reported false positives and no false negative results using the
RapidChek cultural method as the detection system with the food and surfaces
tested (0% false negative rate, 3% false positive rate). In total, the RapidChek
LFD and cultural methods reported 189 and 191 confirmed positive results, while
the reference methods reported 169 confirmed results. The 2 methods have been
shown to be capable of detecting very low levels (1cfu/25g) of Listeria spp. in a
variety of food and has performed as well or better than the reference method with
select environmental matrices. Sample matrix effects on the LFD within the food
and environmental samples examined in these studies were not apparent. Assay
robustness studies indicate that the assay will perform under a wide range of
environmental conditions. The assay is stable for at least a year at room
temperature and results are highly reproducible from lot to lot.
16 Conclusions
16.1The use of the LFD as well as the MOX plate with the 40h proprietary enrichment
method has demonstrated excellent accuracy, sensitivity and specificity throughout
this study. The overall accuracy of the LFD method was 137%, with 99% sensitivity
and 100% specificity. Similarly, the overall accuracy for the RapidChek cultural
method was 138% with 100% sensitivity and 98% specificity. There was excellent
method agreement between the lateral flow system and the cultural confirmation.
Results from select food matrices, specifically, deli turkey, pepperoni, roast beef, hot
dogs, smoked fish, pasteurized whole milk, ice cream, cooked shrimp, potato salad,
and ricotta cheese, indicate that levels as low as 0.2cfu/25g can be successfully
detected although overall, these studies demonstrates that low levels (1-10cfu/25g) of
Listeria can be recovered and detected in a food matrix in a minimum of 40 h.
Similarly, the test methods consistently report greater numbers of confirmed positive
results from stainless steel, painted concrete and rubber environmental samples.
23
17 Reference
17.1 Peccio, A; Autio, T. Kokeala, H., Rosminir, R. and Trevisnai, M. (2003) L.

monocytogenes occurrence and characterization in meat processing plants. Letters in
Applied Microbiology, 37: 234-238.
17.2 Miethtinen, M.K., Palma, L., Bjorkroth, K.J., and Korkeala, H. (2001) Prevalence of
L. monocytogenes in broilers at the abbatoir processing plant and retail level. Journal of
Food Protection, 64, 994-999.
17.3 Cox, L.J., Kleiss, T., Cordier, L., Cordellana, C., Knokel, P., Pedrazzini, C., Beumer,
R. and Siebenga, A. (1989) Listeria species in the food processing, non food and
domestic environments. Food Microbiology, 6: 49-61.
17.4 Schuchat, A; Swaminathan, B. and Broome, C.V. (1991) Epidemiology of human

listeriosis. Clinical Microbiological Reviews, 4: 169-183.
17.5 Doganay, M. (2003) Listeriosis: clinical presentation. FEMS Immunological Medical

Microbiology, 35, 173-175.
17.6 Hitchins, A.D. (1998) US Food Drug and Administration, Bacteriological Analytical
Manual; 8th Edition; Chapter 10: Listeria monocytogenes.
17.7 USDA/FSIS, (2001) Microbiology Laboratory Guidelines, Chapter 8; revision 2;

Isolation and Identification of Listeria monocytogenes from red meat, poultry, egg and
environmental samples.
24
Figure 1: Image of the RapidChek lateral flow device for Listeria.
(a) (b) (c)
Three RapidChek devices are shown, (a) one which has not be used; (b) one which has been
run with a negative cheese sample, displaying one red line indicative of a negative result;
(c) one device which has been run with a positive cheese sample, displaying two red lines
indicative of a positive result. The kits desiccated storage canister is seen to the left of the
photo.
25
Table 1 Listeria Isolates Source List – Inclusivity Study
# Species Serotype Strain Source LFD

Activity
1 L. monocytogenes 1/2a ATCC 51774a Human Blood +
2 L. monocytogenes 1/2a SDI 10-3b/c1b +
3 L. monocytogenes 1/2a SDI 10a-3b/c2b +
4 L. monocytogenes 1/2a SDI 11-3b/c3b +
5 L. monocytogenes 1/2a USDA 472c +
6 L. monocytogenes 1/2b SDI 12-3b/c5b +
7 L. monocytogenes 1/2c SDI 13-3b/c7b +
8 L. monocytogenes 1 ATCC 7644a Human +
9 L. monocytogenes 2 ATCC 19112a Human Spinal +
Fluid
10 L. monocytogenes 3 ATCC 19113a Human +
11 L. monocytogenes 3a SDI 14-3b/c9b +
12 L. monocytogenes 3b SDI 15-3b/d2b +
13 L. monocytogenes 3c SDI 16-3b/d4b +
14 L. monocytogenes 4a SDI 17-3b/d6b +
15 L. monocytogenes 4ab SDI 18-3b/d8b +
16 L. monocytogenes 4b ATCC 13932a Human Spinal +
Fluid
17 L. monocytogenes 4b ATCC 19115a Human +
18 L. monocytogenes 4b ATCC 43256a Mexican Cheese +
19 L. monocytogenes 4b ATCC 51414a Raw Milk +
20 L. monocytogenes 4b SDI 19-3b/e1b +
21 L. monocytogenes 4b U of G H7650d +
22 L. monocytogenes 4c SDI 20-3b/e3b +
23 L. monocytogenes 4d SDI 21-3b/e5b +
24 L. monocytogenes 4e SDI 22-3b/e7b +
25 L. monocytogenes 7 SDI 23-3b/e9b +
26 L. monocytogenes SDI-51b Chicken Isolate +
27 L. monocytogenes U of G H7649d +
28 L. monocytogenes SDI 201b +
29 L monocytogenes SDI-52b Raw Beef +
30 L. grayi ATCC 19120a Chinchilla Feces +
31 L. gray ATCC 25401a Corn Stalks +
32 L. innocua USDA 15-666c +
33 L. innocua 6a ATCC 33090a Cow Brain +
34 L. innocua 6b ATCC 33091a Human Feces +
35 L. innocua SDI-3b Beef Isolate +
36 L. innocua SDI-198b Drain Sponge +
37 L. innocua SDI-53b Raw Turkey +
38 ATCC 51334a Clethrionomys +
L. seeligeri glareolus Intestine
39 L. seeligeri 4a ATCC 51335a +
26
40 L. seeligeri ATCC 35967a Soil +
41 L. seeligeri SDI 3BF1b +
42 L. seeligeri SDI 3BF2b +
43 L. welshimeri 6b ATCC 35897a Plant Material +
44 L. welshimeri 6a ATCC 43548a +
45 L. welshimeri SDI-199b +
46 L. welshimeri SDI-50b Chicken Isolate +
47 L. welshimeri SDI-54b Smoked Salmon +
48 L. ivanovii SDI 200b +
49 L. ivanovii ATCC 700402a +
50 L. ivanovii ATCC 19119a Sheep +
a
American Type Culture Collection, Manassas, VA; b Strategic Diagnostics Inc. Culture Collection, Newark, DE; c Listeria Reference
Laboratory, Donald S. Munro Collection;c; cUnited States Department of Agriculture, Wyndmoor, PA; d University of Georgia Culture
Collection, Athens, GA.
27
Table 2: Non-Listeria isolates
Strain BHI RapidChek Listeria All strains for

1 Brochothrix thermosphacta 11509 - - inclusivity and
2 Citrobacter freundii 7A12 - - exclusivity were
3 Enterobacter cloacae #2 - - typed internally
4 Lactobacillus plantarum 8014 - - according to the
USDA/FIS
5 Micrococcus luteus 533 - -
confirmatory
6 Rhodococcus equi ATCC 7698 - - procedure (10).
7 Salmonella tyhpimurium 14028 - -
8 Streptococcus mitis - -
9 Proteus vulgaris ATCC 6380 - -
10 Bacillus cereus 11778 - -
11 Enterococcus faecalis 19433 - -
12 Staphylococcus aureus + -
13 Acinetobacter baumannii 19606 - -
14 Aeromonas hydrophila #10 - -
15 Chryseobacterium meningosepticum 13253 - -
16 Klebsiella pneumoniae #9 - -
17 Pseudomonas aeruginosa 10145 - -
18 Myroides odoratus 4651 - -
19 Vibrio spp. 62A1 - -
20 Yersinia enterocolitica 23715 - -
21 Lactobacillus/coccus lacti 11454 - -
22 Lactobacillus acidophilus 314 - -
23 Pseudomonas spp. - -
24 E. coli 0157 35150 - -
25 Hafnia alvei ATCC 25927 - -
26 Enterococcus durans biotype 6 - -
27 Enterococcus faecalis Biotype 43497 - -
28 Corynebacterium diptheriae biotype 13 - -
29 Enterococcus faecium biotype 11 - -
30 Corynebacterium pseudogenitalium biotype 2997 - -
31 Enterococcus raffinosus biotype 5 - -
32 Leuconostoc citreum biotype 109929 - -
33 Streptococcus pyogenes 19615 - -
34 Staphylococcus vitulinus biotype 265308 - -
35 Bacillus subtilits biotype 987649 - -
36 Corynebacterium spp. Biotype 5 - -
28
Table 3: Inclusivity and Exclusivity Study
Organism Media type Total number of Total positive on Sensitivity/

strains tested on RapidChek Specificity
RapidChek LFD (%)
Listeria species RapidChek 50 50 100
BHI 30 1 97
Non Listeria species
RapidChek 30 0 100
29
Table 4:Extramural Comparison of the 40-h RapidChek Listeria Methods with the USDA/FSIS and FDA/BAM Reference Methods: Roast Beef,
Ricotta Cheese.
Sample Method type Strain MPN # of Presumptive Confirmed Sensitivity Specificity Method Chi-
Matrix /25g Sample Positives Positives (%) (%) Agreement Square
(%)
Roast Beef RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
LFD innocua
0.225 20 14 14 100 100 75 1.45
40 h (0.05-0.93)*
0 5 0 0 n/a 100 100 n/a
RapidChek
Cultural
0.225 20 14 14 100 100 75 1.45
40 h
(0.05-0.93)*
0 5 0 0 n/a n/a n/a n/a
USDA/FSIS
48 h 0.225 20 9 9 n/a n/a n/a n/a
(0.05-0.93)*
Ricotta RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
Cheese LFD monocytogenes
40 h 4ab 0.575 20 12 12 100 100 95 0
(0.17-2.0)*
RapidChek 0 5 0 0 n/a 100 100 n/a
Cultural
40 h 0.575 20 12 12 100 100 95 0
(0.17-2.0)*
FDA/BAM 0 5 0 0 n/a n/a n/a n/a
48 h
0.575 20 13 13 n/a n/a n/a n/a
(0.17-2.0)*
n/a = not applicable
Note: Roast Beef Aerobic Plate Count = 1.0 X 10^4 cfu/g
Ricotta Cheese Aerobic Plate Count = <10 cfu/g
* = 95% confidence Interval
1
Table 5: Extramural Comparison of the 40-h RapidChek Listeria Methods with the USDA/FSIS Reference Method: Rubber environmental samples.
Sample Method type Strain Nominal cfu # of Presumptive Confirmed Sensitivity Specificity Method Chi-
Matrix /4 inch2 Sampl Positives Positives (%) (%) Agreement Square
e (%)
Rubber Listeria 0 5 0 0 n/a 100 100 n/a
Surfaces RapidChek monocytogenes
LFD 4ab 20 9 9 100 100 65 4.0
40 h
1100
0 5 0 0 n/a 100 100 n/a
RapidChek
Cultural 20 9 9 100 100 65 4.0
1100
0 5 0 0 n/a n/a n/a n/a
USDA/FSIS
48 h 20 3 2 n/a n/a n/a n/a
1100
2
Table 6: Intramural Comparison of the 40-h RapidChek Listeria Methods with the USDA/FSIS Reference Method: Deli
Turkey, Hotdogs.
(%)
Deli RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
Turkey LFD monocytogenes
40 h 1/2a 0.4 20 14 15 93 100 95 0
(0.1-1.3)*
RapidChek 0 5 0 0 n/a 100 100 n/a
Cultural
40 h 0.4 20 15 15 100 100 90 0.1
(0.1-1.3)*
USDA/FSIS 0 5 0 0 n/a n/a n/a n/a
48 h
0.4 20 13 13 n/a n/a n/a n/a
(0.1-1.3)*
Hotdogs RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
LFD welshimeri
40 h 2.3 20 16 16 100 100 100 0.13
(0.6-9.5)*
RapidChek 0 5 0 0 n/a 100 100 n/a
Cultural
40 h 2.3 20 16 16 100 100 100 0.13
(0.6-9.5)*
48 h
2.3 20 16 16 n/a n/a n/a n/a
(0.6-9.5)*
Note: Deli Turkey Aerobic Plate Count = <9 X 10^3 cfu/g
Hotdogs Aerobic Plate Count = < 8 10^3 cfu/g
Table 7: Intramural Comparison of the 40-h RapidChek Listeria Methods with the USDA/FSIS and FDA/BAM Reference
Methods: Pepperoni and Smoked Fish.
Pepperoni RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
LFD monocytogenes
40 h 3b 1.1 20 10 10 100 100 90 0.07
(0.3-4.3)*
RapidChek 0 5 0 0 n/a 100 100 n/a
Cultural
40 h 1.1 20 10 10 100 100 90 0.07
(0.3-4.3)*
48 h
1.1 20 12 12 n/a n/a n/a n/a
(0.3-4.3)*
Smoked RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
Fish LFD monocytogenes
40 h 7 5.0 20 14 14 100 100 90 0.1
(1.5-17.8)*
RapidChek 0 5 0 0 n/a 100 100 n/a
Cultural
40 h 5.0 20 14 14 100 100 90 0.1
(1.5-17.8)*
48 h
5.0 20 16 16 n/a n/a n/a n/a
(1.5-17.8)*

Note: Pepperoni Aerobic Plate Count = 1.8 X 10^5 cfu/g
Smoked Fish Aerobic Plate Count = 1.89 X 10^5 cfu/g
4
Table 8: Intramural Comparison of the 40-h RapidChek Listeria Methods with the FDA/BAM Reference Method: Cooked
Shrimp, and Potato Salad.
(%)
Cooked RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
Shrimp LFD monocytogenes
40 h 3c 0.2 20 11 11 100 100 100 0.1
(0.1-0.9)*
RapidChek 0 5 0 0 n/a 100 100 n/a
Cultural
40 h 0.2 20 11 11 100 100 100 0.1
(0.1-0.9)*
48 h
0.2 20 11 11 n/a n/a n/a n/a
(0.1-0.9)*
Potato RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
Salad LFD monocytogenes
40 h 1/2b 1.1 20 16 17 94 100 90 0.17
(0.2-4.3)*
RapidChek 0 5 0 0 n/a 100 100 n/a
Cultural
40 h 1.1 20 18 17 100 87 95 0
(0.2-4.3)*
48 h
1.1 20 18 18 n/a n/a n/a n/a
(0.2-4.3)*

Note: Cooked Shrimp Aerobic Plate Count = 1.8 X 10^4 cfu/g
Potato Salad Aerobic Plate Count = 1.8 X 10^4 cfu/g
Table 9: Intramural Comparison of the 40-h RapidChek Listeria Method with the FDA/BAM Reference Method: Whole Milk,
and Ice cream.
(%)
Whole RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
Milk LFD monocytogenes
40 h 4b 5.3 20 17 17 100 100 95 0
(1.5-18.3)*
RapidChek 0 5 0 0 n/a 100 100 n/a
Cultural
40 h 5.3 20 17 17 100 100 95 0
(1.5-18.3)*
48 h
5.3 20 18 18 n/a n/a n/a n/a
(1.5-18.3)*
Ice Cream RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
LFD monocytogenes
40 h 3a 5.8 20 20 20 100 100 80 2.25
(1.7-20.3)*
RapidChek 0 5 0 0 n/a 100 100 n/a
Cultural
40 h 5.8 20 20 20 100 100 80 2.25
(1.7-20.3)*
48 h
5.8 20 16 16 n/a n/a n/a n/a
(1.7-20.3)*

Note: Whole Milk Aerobic Plate Count = 9 X 10^3 cfu/g
Ice Cream Aerobic Plate Count = < 9 X 10^3 cfu/g
6
Table 10: Intramural Comparison of the 40-h RapidChek Listeria Methods with the USDA/FSIS Reference Method: Painted
Concrete, and Stainless Steel.
Sample Method type Strain Nominal # of Presumptive Confirmed Sensitivity Specificity Method Chi-
Matrix MPN Sample Positives Positives (%) (%) Agreement Square
/inch2 (%)
Painted RapidChek Listeria 0 5 0 0 n/a 100 100 n/a
Concrete LFD monocytogenes
40 h 4b 1X104 20 18 18 100 100 60 6.13
RapidChek 0 5 0 0 n/a 100 100 n/a

Cultural
40 h 1X104 20 19 18 100 86 60 6.13

48 h
1X104 20 10 10 n/a n/a n/a n/a
Stainless RapidChek Listeria 0 5 0 0 n/a 100 100 n/a

Steel LFD innocua
40 h 5.5X102 20 18 18 100 100 82 0.57
RapidChek 0 5 0 0 n/a 100 100 n/a

Cultural
40 h 5.5X102 20 18 18 100 100 82 0.57

48 h
5.5X102 20 15 15 n/a n/a n/a n/a
7
Table 11: Assay Temperature and Read Time Latitude Study
Operator Inoculum Number Reading Number of RapidChek Positive
Level of of Time of LFD Results at
Listeria Samples Device Various Temperatures/Total Tested
innocua Tested (minutes)
4 oC 25 oC 45 oC
1 0 cfu/inch2 5 5 0/5 0/5 0/5
(negative) 8 0/5 0/5 0/5
10 0/5 0/5 0/5
15 0/5 0/5 0/5
20 0/5 0/5 0/5
555 cfu/inch2 10 5 10/10 10/10 10/10

8 10/10 10/10 10/10
10 10/10 10/10 10/10
15 10/10 10/10 10/10
20 10/10 10/10 10/10
2 0 cfu/inch2 5 5 0/5 0/5 0/5

(negative) 8 0/5 0/5 0/5
10 0/5 0/5 0/5
15 0/5 0/5 0/5
20 0/5 0/5 0/5
555 cfu/inch2 10 5 10/10 10/10 10/10

8 10/10 10/10 10/10
10 10/10 10/10 10/10
15 10/10 10/10 10/10
20 10/10 10/10 10/10
3 0 cfu/inch2 5 5 0/5 0/5 0/5

(negative) 8 0/5 0/5 0/5
10 0/5 0/5 0/5
15 0/5 0/5 0/5
20 0/5 0/5 0/5
555 cfu/inch2 10 5 10/10 10/10 10/10

8 10/10 10/10 10/10
10 10/10 10/10 10/10
15 10/10 10/10 10/10
20 10/10 10/10 10/10
Table 12: Assay Sample Volume Latitude Study
Operator Sample RapidChek Positive RapidChek Positive Accuracy
Volume Results from Non- Results from (%)
(uL) Inoculated Samples Inoculated Samples
1 300 0/5 10/10 100

400 0/5 10/10 100
500 0/5 10/10 100
2 300 0/5 10/10 100

400 0/5 10/10 100
500 0/5 10/10 100
3 300 0/5 10/10 100

400 0/5 10/10 100
500 0/5 10/10 100
10
Table 13: An evaluation of 20 spiked and 5 non-inoculated smoked fish samples with 3
different lots of RapidChek Lateral Flow Devices.
Sample Inoculum
Confirmation
ID Level Device Lots
Result
R&D
(cfu/25g) MFG 4a1043-3 MFG 4b1045-x
Nb1987:72
1 5 + + + +
2 5 + + + +
3 5 + + + +
4 5 + + + +
5 NI - - - -
6 NI - - - -
7 5 - - - -
8 5 + + + +
9 5 - - - -
10 5 + + + +
11 5 + + + +
12 NI - - - -
13 5
- - -
-
14 + + +
5 +
15 - - -
5 -
16 + + +
NI +
17 - - -
18 5 - - - -
19 5 - - - -
20 NI - - - -
21 5 + + + -
22 5 + + + +
23 5 + + + +
24 5 + + + +
25 5 + + + +
NI = non-inoculated sample
11
Table 14: Assay Accelerated Stability Study
Time Strip Lot Calibrator RapidChek LFD Results at Various Storage
(days) Level Temperatures
o
4 C 25 oC 37 oC 45 oC
0 R&D 0 - - - -
Nb1987:72 Low + + + +
High + + + +
MFG 0 - - - -
4a1043-3 Low + + + +
High + + + +
R&D
20 Nb1987:72 0 - - - -
Low + + + +
High + + + +
MFG
4a1043-3 0 - - - -
Low + + + +
R&D High + + + +
Nb1987:72
35 0 - - - -
Low + + + +
MFG High + + + +
4a1043-3
0 - - - -
Low + + + +
High + + + +
12
Table 15: RapidChek Media Preparation Latitude Study
Inoculum Background Media Treatment
Food Species cfu/25g cfu/g Rehydrated Autoclaved
Potato Salad L.mono 1/2b 2.8 1.8E4 17 16
Smoked Fish L.mono 7 3 7.47E5 18 17
Total 35 33
Note: n = 20 for each food type
Table 16: RapidChek Sample Preparation Latitude Study

Live 5 Minute 10 Minute 15 Minute 30 Minute
L. mono 1/2a + + + + +
L. mono 1/2b - + + + -
L. mono 1/2d - + + + +
L. mono 3a + + + + +
L. mono 3b - + + + +
L. mono 4a - + + + +
L. mono 4b + + + + +
L. mono 4c - + + + +
L. mono 4d - + + + +
L. innocua 6b - + + + +
L. welshimeri 6a - + + + +
L. mono 3c + + + + +
L. mono 4ab - + + + +
L. mono 7 - + + + +
Peptone Water - - - - -
Note: All samples ran at 1E6.
13
Table 17 Media volume latitude study with sponge samples from stainless steel
environmental surfaces inoculated with L. innocua and co-inoculated with growth
competitors .
Sample Type Inoculation Level 100mL RapidChek 60mL RapidChek
Cfu/4 inch2 Enrichment Volume Enrichment Volume
Stainless steel Non-inoculated 0/5 0/5
w/sponge 1.3x102 9/10 10/10
2.2x102 10/10 10/10
6.5x102 10/10 10/10
14

Laporan Validasi Listeria-AOAC

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Laporan Validasi Listeria-AOAC

Uploaded by

Copyright:

Available Formats

RapidChek for the Detection of Listeria species in a Variety of Foods and

Select Environmental Surfaces

Strategic Diagnostics Inc., 111 Pencader Drive, Newark, Delaware 19702

1.1 Target organisms – Listeria species

2.1 Sensitivity – Rate of sensitivity = (No. of LFD Test Positives)/(No. Confirmed

2.2 Specificity – Rate of specificity = (No. of LFD Test Negative)/(No. Confirmed

2.4 Accuracy – (No. of Confirmed Test Positives/Total No. Reference Positives) ×

4.0 General Information

In order to prevent occurrence of listeriosis, government regulations stipulate the total

5.0 Test Kit Information

5.2 Catalog Number – 7000171

6.1 RapidChek Listeria Lateral Flow assay

6.2.2 Buffered Listeria Enrichment Broth Base (BLEB) - (Oxoid).

sodium pyruvate, 110mg/L

Dissolve 47g of powder media in one liter of sterile distilled or deionized

6.2.3 BLEB Supplement (selective agents):

add 47g of powder media to 1L of deionized water. Autoclave for 15

6.2.5 Fraser Broth – (Difco Becton Dickinson)

casien peptone, 5.0g/L

6.2.6 Dey-Engley neutralizing broth (D/E) – (Difco)

add 39g of powder media to 1L of deionized water. Autoclave for 15

Supplements: (Difco Becton Dickinson)

add 57.5g of powder media to 1L of deionized water. Autoclave for 15 minutes at

6.3 Disposable 200 µl pipette tips

6.4 Filtered stomacher bags

6.5 10uL and 1uL sterile, disposable loops (FisherBrand)

6.6 Vortex mixer

add 57.5g of powder media to 1L of deionized water. Autoclave for 15 minutes at

6.9 Motility Test Media (MTM)

beef extract 3g/L

6.10 API Listeria ID Kit – (bioMerieux)

6.11 Hydrogen peroxide solution, 3%

6.12 Gram stain reagents (Fisher Scientific)

6.13 Immersion Oil

6.14 Microscope slides, glass

6.15 Cover slips, glass

6.16 Non-bacteriocidal 7.5x4x0.3cm sterile cellulose sampling sponge pre-moisten

6.17 Sterile cotton-tipped swab – (Fisher)

7.2 Micropipet – Capable of accurately dispensing 400 µl.

7.6 Top loading balance – To weigh 25 g ( + 0.1g) test samples.

7.7 Zeiss phase contrast microscope.

8.0 Standard Reference Material

8.2 Chapter 10 entitled “Detection and Enumeration of Listeria monocytogenes in

9.0 Safety Precautions

10.0 Sample Preparation

10.1 Food Samples

10.1.1 Aseptically weigh a representative 25 g sample and add to filtered

10.1.3 Stomach samples for approximately 1-2 minute.

10.1.5 Incubate USDA (UVM) samples at 30+/-2oC for 22 +/-2 h in a stationary

10.1.6 Incubate Proprietary Media samples at 30oC for 40 h in a stationary

10.2 Isolation and Confirmation Procedure

10.2.2 BAM Reference Method: After 24 and 48 h streak BLEB enrichment to

10.2.3 FSIS Reference Method: After 24 and 48 h streak UVM and FB

11.0 Set Up and Operation

11.1 Label each tube with the appropriate sample identification.

11.6 Insert test strip and let develop for 10 minutes.

13.0 Independent Validation Studies

13.1 Independent validation studies were conducted by Silliker Laboratories in South

13.2.1.1 Roast Beef

L. innocua (ATCC 33090) was grown in 10 ml TSB

Inoculated meat was divided into two sets of twenty 25

A second set of twenty 25 g samples of inoculated

All samples were confirmed using the standard cultural

13.2.1.2 Ricotta Cheese