Strategic Diagnostics Inc
RapidChek™ Listeria species Test User Guide
RapidChek
Listeria species Test Kit (45-Strips)
Part Number : 7000171-7000175*
captures the Listeria–antibody–gold complex,
causing the formation of an antibody–Listeria
“sandwich” and development of red color at the
test line. Antibody–Listeria sandwiches are not
formed in the absence of Listeria, resulting in no
red color development at the test line. Reagents
immobilized at the control line capture excess
gold reagent passing through the test line. The
presence of red color at the control line indicates
that the strip test flowed correctly. Therefore, the
presence of only one line (control line) on the
membrane indicates a negative sample and the
presence of two lines indicates a positive sample.
Intended Use
The RapidChek Listeria Lateral Flow Test Kit is
designed to detect the Listeria species in a variety
of ready to eat foods (deli turkey, pepperoni, hot
dogs, roast beef, potato salads), dairy (ice cream,
soft cheese, milk), fish products (cooked shrimp
and smoked fish) and on environmental surfaces
(including rubber, painted concrete and stainless
steel). The test kit permits the presumptive
detection and identification of the target pathogen
by a one step process in a minimum of 40 hours
when present at levels of one Listeria organism
per 25 grams of sample. The RapidChek Listeria
test kit is designed to be used by trained
technicians. Although, the device is easy to use,
the protocol involves the use of potentially
hazardous microorganisms and appropriate safety
must be practiced.
*Contents of Kits
Part Number 7000171(Kit only)
Description
RapidChek Listeria strips
Transfer pipettes (400 L)
Plastic tubes (12 x 75mm)
User Guide
Principle of the Assay
This immunoassay test uses a double antibody
sandwich format. An antibody specific to Listeria
is sprayed and immobilized in a line on the surface
of a membrane comprising a “test line”. A second
antibody reagent, also recognizing Listeria and
labeled with colloidal gold, is contained within a
reagent pad upstream from the test line on the
membrane. As the sample moves by capillary
action from the filter pad into the antibody–gold
pad, the antibody–gold reagent specifically binds
Listeria and moves with the liquid sample into the
test membrane. The sample passes through the test
line where the immobilized Listeria antibody
Part # 7000171-175
Quantity
45
45
45
1
Part Number 7000172 (Media) & 7000173
(supplement)
Description
Quantity
RapidChek Listeria media
500g
RapidChek Listeria supplement
10g
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Part Number 7000174 (Environmental Kit)
Description
Quantity
RapidChek Listeria strips
45 x 2
Transfer pipettes (400 L)
90
Plastic tubes (12 x 75mm)
90
RapidChek Listeria media
500g
RapidChek Listeria supplement
10g
User Guide
1
Part Number 7000175 (Food Kit)
Description
RapidChek Listeria strips
Transfer pipettes (400 L)
Plastic tubes (12 x 75mm)
RapidChek Listeria media
RapidChek Listeria supplement
User Guide
Quantity
45
45
45
500g
10g
1
Materials Required but not supplied
Stomacher-type bags or equivalent (filtered)
Stomacher machine (Stomacher 400 Circulator,
Seward, available from Brinkmann Inc, Westbury,
NY 11590) or equivalent
Plastic test tube rack (Fisher Scientific,
dimensions 20 cm x 10 cm, holds 75 x 12mm
tubes)
Hotplate (capable of reaching 100C)
Stainless Steel dish/Pyrex dish (40 cm x 15 cm)
Balance capable of weighing 25 -1000 g with
sensitivity of 0.2g.
Incubator capable of maintaining 30C  0.5C
40 Hour RapidChek Procedure
RapidChek Listeria Media (Part # 7000172),
500g
RapidChek Listeria media supplement (Part #
7000173), 10.0g
Sample Preparation and Enrichment
For RapidChek Media System
A. Media Preparation
1. Sterilize one liter of water either by
autoclaving or filtration (filter with pore size
of 0.2m) into a sterile container.
2. Equilibrate the sterilized water to 20-30C.
3. Weigh 53.0  0.2g of RapidChek Listeria
Media, and 1.0g +/- 0.05g of RapidChek
Listeria media supplement, and add to the
sterilized water which has been equilibrated to
30C. Shake vigorously until the media is
completely mixed.
4. Rehydrated media should be used within 5
hours of preparation at room temperature or
24h at 4C. For best results, use the media as
soon as it is prepared.
ALTERNATIVELY, the RapidChek media can
be AUTOCLAVED.
1a. Add 53.0g  0.2g of RapidChek Listeria
Media base to 1 litre of room temperature
distilled water, to ensure full dissolution of
solids and mix at 20-30C until dissolved
completely.
2a. Autoclave at 121C for 15 minutes.
3a. Allow the RapidChek Listeria media to cool to
room temperature. Just prior to use, add 1.0g
+/- 0.05g of RapidChek Listeria media
supplement to the media base.
Note: The media base can be stored at 4C for up to two
weeks. After refrigeration, media should be equilibrated
to 20-30C before use.
Preparation and Storage of Reagents
The RapidChek Listeria Test Kit should be stored
at room temperature (15C-30C). The
RapidChek Listeria strips used in this kit must be
kept in the canister with the indicating desiccant.
The indicating desiccant should be blue in color.
After opening the canister, care should be taken to
Part # 7000171-175
re-seal the closure to protect the strips from
moisture.
B. Sample Enrichment
1. Add 25g of the sample to be analyzed into the
sterile Stomacher bag.
2. Add 225 mL of RapidChek media (prewarmed to 30C) to the Stomacher bag
containing the sample.
Note: For environmental samples, sponges are placed in
100ml of RapidChek Listeria broth and proceed as with
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other samples enrichments. Recommended sponge types
include cellulose non bactericidal sampling sponges
available from Solar Biologicals, sponge dimensions are
7.5 x 4 cm, and are premoistened with 10 ml of DE broth.
4 inch square surface is sampled by wiping the sponge on
the surface in a backward and forward motion for 30
seconds. Sponges are then placed in a sterile bag, sealed
and transported at room temperature to the laboratory.
For swabs samples, sterile cotton tipped swabs available
from Fisher Scientific are recommended and these are
pre-moistened in DE broth. One inch square of the
surface is sampled by rubbing the swab in a backward
and forward motion for 30 seconds. Swabs are then
places in a sterile bag, which is sealed and transported to
the laboratory at room temperature conditions. Swabs
are placed in 10 ml of RapidChek broth and processed as
previously described.
3. Place the sample bag into a Stomacher device
and stomach for 30 seconds.
4. Close the bag loosely and incubate for 40
hours at 30C 0.5C.
Note: Using RapidChek media, samples can be enriched
for as short as 40 hours. Proceed to RapidChek Listeria
test procedure for detection of organisms.
5.
Proceed to RapidChek Listeria test procedure
for the detection of organisms.
RapidChek Listeria Test Procedure
1. Take one transfer pipette from the bag (or
utilize calibrated pipette capable of dispensing
400l). Squeeze and hold the bubble on top of
the pipette and place in the sample enrichment.
2. Release the bulb completely filling the barrel
of the pipette.
Note: The bubble will not completely fill with solution.
3. Transfer aliquot of enriched broth to plastic
tubes supplied in a test tube rack.
4. Place tubes in a test tube rack in a boiling
water bath (100C) for 5 mins.
Note: Samples can be boiled for a minimum of 5 minutes
and a maximum of 15 minutes.
5. Remove tubes after boiling step and allow to
cool to room temperature prior to testing.
Part # 7000171-175
6. Remove the required number of test strips
from the canister and label each strip with the
appropriate sample identification.
7. Insert the strip with arrows facing
downwards into the tube.
8. Let the strip develop for 10 minutes.
9. The appearance of one red line (control) on
the strip indicates a negative result.
10. The appearance of two red lines on the strip
indicates a positive result.
Check the result at ten (10) minutes after adding
strip to the sample. At least one line, the Control
Line, should always develop. A red line in this
position indicates that the strip is functioning
properly. A red line appearing below the Control
Line is the Test Line and indicates a positive
result. If the test strip displays two (2) red lines,
the test is complete and the sample is positive for
Listeria.
If at 10 minutes the test strip only shows a clearly
visible Control Line, then the sample is negative
for Listeria. If no control line develops within 10
minutes, the test is invalid and needs to be
repeated.
Note: Test strip results should be interpreted
after 10 minutes. Test strips interpreted after 20
minutes are invalid.
Confirmation
Presumptive positive sample results must be
confirmed by Bacteriological Analytical Manual
(BAM) or the USDA/FSIS methods for the
detection of Listeria. It is recommended that roast
beef, deli turkey, hot dogs, pepperoni, and all
environmental samples be confirmed using the
FSIS/USDA protocol. It is recommended that
ricotta cheese, smoked fish, cooked shrimp, whole
milk, ice cream, and potato salad be confirmed
using the FDA protocol.
Enriched media samples used in the Listeria Test
Procedure prior to boiling can be used for this
confirmation. For confirmation procedure see the
following:
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(1) FDA/BAM – Detection and Enumeration
of Listeria (chapter 10) In: US Food, Drug
and Administration , Center for Food
Safety and Applied Nutrition,
Bacteriological Analytical Manual:
website location:
http://www.cfsan.fda.gov/~ebam/bam10.html
(2) USDA/FSIS – Isolation and Identification
of Listeria monocytogenes from red meat,
poultry, egg and environmental samples.
(Chapter 8, revision 2): In : Microbiology
Laboratory Guidelines, USDA-FSISOPHS; website location:
http://www.fsis.usda.gov/ophs/microlab/ml
g8.03.pdf
For all procedures, positive and negative controls
for enrichment, detection and confirmation are
recommended as part of good laboratory practice.
ability of the target pathogen to adequately reach the test’s
detection level in the enrichment media. This ability may be
influenced by a variety of factors including but not limited to ;
competitive flora, sample matrix, sample size and condition of
the target pathogen.
Disposal
Decontaminate RapidChek test strips, pipettes and
media by autoclave, bleach, etc., in accordance
with local, state and federal regulations.
Positive Controls
SDI recommend two controls for use with the
RapidChek Listeria kit:
(1) For RapidChek complete enrichment/detection
system – L. monocytogenes ATTC 19115,
available from American Type Culture Collection
at www.atcc.org
(2) For RapidChek Lateral Flow Strip – Listeria,
genus specific positive control available from
Kirkguard Perry Laboratories at www.KPL.com
Commodities
The RapidChek Listeria Test Kit has been
validated to detect one Listeria cell per 25 grams
of sample. The applicable sample matrices are
ready to eat foods and environmental samples
Please contact SDI Customer Service for specific
applications.
Product Shelf life
The expiration date for the product is displayed
along with the part and lot number on the Product
Label located on the resealable canister. This
product has a 1 year shelf life from the date of
manufacture under desiccated room temperature
(15C-30C) conditions. Contact customer
service with any questions about product shelf life.
Illustration of Positive and Negative Results
Precautions
1. Listeria monocytogenes is a significant human
pathogen. Immuocompromised individuals,
such as pregnant women should not be in the
vicinity of samples being enriched or tested for
Listeria as they represent particularly
susceptible populations. Extreme care should
be used in handling samples which could
potentially contain this pathogen, enriched
media and used test strips. Ensure all
biohazardous waste is disposed of
appropriately.
2. If polypropylene bottles are used for sample
enrichment instead of Stomacher bags, the
bottles should be lined with a disposable
plastic bag to eliminate potential protein
Red Control Line
(Assay has Run Properly)
Red Test Line
(Sample is Positive)
Positive
Negative
Note: Although this test kit system is capable of detecting
target pathogen present in enrichment media at the detection
level sensitivity of the test strip, the successful detection of the
target pathogen in a specific food matrix is dependent upon the
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3.
4.
5.
6.
7.
8.
carryover, which will produce erroneous
results.
Use rehydrated RapidChek, media within 5
hours of preparation. For best results use the
media as soon as it is prepared.
Sample bags should be closed loosely to allow
air exchange during sample enrichment and
optimize pathogen growth and antigenic
expression.
Test strip results should be interpreted after 10
minutes. Test strips interpreted after 20
minutes are invalid.
Storage conditions higher than room
temperature may adversely affect performance
of the test strip.
Do not use test strips beyond the expiration
dating on the kit package label.
Follow standard Good Microbiological
Practices where appropriate.
For Technical & Customer Service call:
Strategic Diagnostics Inc
111 Pencader Drive
Newark, DE 19702
Phone: 800-544-8881
Fax:
302-456-6782
e-mail: techservice@sdix.com
Warranties and Liabilities
Strategic Diagnostics Inc. (“SDI”) warrants the Products
manufactured by it will be free of defects in materials and
workmanship when used in accordance with the applicable
instructions for a period equal to the shorter of one year from
date of shipment of the Product(s) or the expiration date
marked on the Product packaging. Application protocols
published by SDI are intended to be only guidelines for the
Buyers of the Products. Each Buyer is expected to validate
the applicability of each application protocol to in their
individual applications.
SDI MAKES NO OTHER
WARRANTY, EXPRESSED OR IMPLIED. THERE IS
NO WARRANTY OF MERCHANTABILITY OR
FITNESS FOR A PARTICULAR PURPOSE.
SDI's sole obligation with respect to the foregoing
warranties shall be, at its option, to either replace or to
refund the purchase price of the Product(s) or part thereof
that proves defective in materials or workmanship within the
warranty period, provided the customer notifies SDI
promptly of any such defect. SDI SHALL NOT BE
LIABLE FOR ANY DIRECT, INDIRECT OR
CONSEQUENTIAL DAMAGES RESULTING FROM
ECONOMIC LOSS OR PROPERTY DAMAGES
SUSTAINED BY BUYER OR ANY CUSTOMER
FROM THE USE OF THE PRODUCT (S).
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