BIOMÉRIEUX

30122 14080 O - en - 2018/02

VIDAS® UP E. coli O157 (including H7) (ECPT)

For microbiological control only

Based on the specific phage capture technology, VIDAS® UP E. coli O157 (including H7) is an automated qualitative test
®
for use on the VIDAS family of instruments for the detection of potentially enterohemorrhagic E. coli O157 in food
products, soil and environmental samples.

SUMMARY AND EXPLANATION The Solid Phase Receptacle (SPR®) serves as the solid
Escherichia coli O157:H7 is a recognized cause of phase as well as the pipetting device. The interior of the
®
hemorrhagic colitis and has been implicated in a number SPR is coated with recombinant phage tail fiber protein
of outbreaks in Japan, the United States, Canada, the for the capture of E. coli O157 including H7. Reagents for
United Kingdom and Belgium (1, 2). The most common the assay are ready-to-use and pre-dispensed in the
source of infection has been raw beef although other sealed reagent strips.
foods such as milk, apple cider, fresh salad, and spinach All of the assay steps are performed automatically by the
have also been implicated. instrument. The reaction medium is cycled in and out of
®
Infection by E. coli O157:H7 is characterized by severe the SPR several times.
abdominal cramps and bloody diarrhea. Infection Part of the enrichment broth is dispensed into the reagent
progresses to acute kidney failure (hemolytic uremic strip. The E. coli O157 including H7 present are captured
syndrome) in about 10% of the cases. Children under the by the recombinant phage protein coating the interior of
®
age of five and the elderly are the most susceptible (3). the SPR . Unbound components are eliminated during
Thanks to an innovative technology based on a the washing steps. Alkaline phosphatase conjugate is
®
recombinant phage tail fiber protein, VIDAS ECPT
® then cycled in and out of the SPR and will bind to any E.
enables the specific detection of E. coli O157 including coli O157 including H7 which are themselves bound to the
®
H7. phage protein on the SPR wall.
Recent studies have shown that non-H7 E. coli O157 Further wash steps remove unbound conjugate.
strains can be pathogenic since they are carriers of During the final detection step, the substrate (4-Methyl-
®
virulence genes (4, 5, 6) umbelliferyl phosphate) is cycled in and out of the SPR .
The conjugate enzyme catalyzes the hydrolysis of this
PRINCIPLE substrate into a fluorescent product (4-Methyl-
® umbelliferone), the fluorescence of which is measured at
VIDAS ECPT is an automated assay based on the ELFA 450 nm.
technique (Enzyme-Linked Fluorescent Assay) for use on At the end of the assay, the results are analyzed
®
the instruments of the VIDAS family (see the User automatically by the instrument which generates a test
Manual). value for each sample. This value is compared to a set of
stored standards (thresholds) and each result is
interpreted (positive, negative).

CONTENT OF THE KIT (30 TESTS)

30 ECPT Strips STR Ready-to-use.
® ®
30 ECPT SPR s SPR Ready-to-use.
®
Interior of SPR s coated with recombinant tail fiber phage protein.
ECPT Standard S1 Ready-to-use.
(1 x 6 mL) Purified and inactivated membrane extract + preservative + protein stabilizers.
MLE data indicate the confidence interval in Relative Fluorescence Value
("Standard (S1) RFV Range").
ECPT Positive Control C1 Ready-to-use.
(1 x 6 mL) Purified and inactivated membrane extract + preservative + protein stabilizers.
MLE data indicate the confidence interval in test value ("Control C1 (+) Test Value
Range").
Negative Control C2 Ready-to-use.
(1 x 6 mL) TRIS Buffered Saline (TBS) (150 mmol/L) - Polysorbate 20 pH 7.6 + preservative.
MLE data indicate the maximum acceptable value in Test Value ("Control C2 (-)
Test Value Range").
Specifications for the factory master data required to calibrate the test:
 MLE data (Master Lot Entry) provided in the kit.
or
 MLE bar code printed on the box label.
1 Package Insert downloadable from www.biomerieux.com/techlib

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VIDAS UP E. coli O157 (including H7) (ECPT) 14080 O - en - 2018/02

The SPR The Reagent Strip

The interior of the SPR® is coated during production with The strip consists of 10 wells covered with a labeled foil
recombinant tail fiber phage protein. seal. The label comprises a bar code which mainly
®
Each SPR is identified by the "ECPT" code. Only remove indicates the assay code, kit lot number and expiration
®
the required number of SPR s from the pouch and date.
carefully reseal the pouch after opening. The foil of the first well is perforated to facilitate the
introduction of the sample.
The last well of each strip is a cuvette in which the
fluorometric reading is performed.
The wells in the center section of the strip contain the
various reagents required for the assay.
Description of the ECPT strip
Well Reagents
1 Sample well: dispense 0.5 mL of enrichment broth, standard or control.
2 Pre-wash buffer (400 µL): TRIS Buffered Saline (TBS) (150 mmol/L) - Polysorbate 20 pH 7.6 +
preservative.
3-4-5-7-8-9 Wash buffer (600 µL): TRIS Buffered Saline (TBS) (150 mmol/L) - Polysorbate 20 pH 7.6 +
preservative.
6 Conjugate (400 µL): Alkaline phosphatase + preservative.
10 Reading cuvette with substrate (300 µL): 4-Methyl-umbelliferyl-phosphate (0.6 mmol/L) +
diethanolamine* (DEA) (0.62 mol/L or 6.6%, pH 9.2) + preservative.

* Signal Word: DANGER

Hazard statement
H318 : Causes serious eye damage.
Precautionary statement
P280: Wear protective gloves/protective clothing/eye protection/face protection.
P305 + P351 + P338: IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present
and easy to do. Continue rinsing.
For further information, refer to the Material Safety Data Sheet.

REAGENTS, MATERIALS AND DISPOSABLES The following references, available from bioMérieux or
REQUIRED BUT NOT PROVIDED one of its companies, are given as a guide.
®
 Instrument of the VIDAS family  Buffered Peptone Water
 Disposable pipettes and/or micropipettes to dispense - 5-liter bag (ref. AEB910305/2)
appropriate volumes - 3-liter bag (ref. AEB910303/4)
 Swabs/sponges for environmental sampling - 225 mL bottle (ref. 42043)
®
 VIDAS Heat and Go (bioMérieux ref. 93554 or 93555 - 225 mL mini-bag (ref. 42729)
or 93556: Contact your bioMérieux representative) or a - 90 mL bottle (ref. 42042)
water-bath (95-100°C) or an equivalent system - 9 mL tube (ref. 42111)
 Paddle blender (example: SMASHER™ ref.  Cefixime-Tellurite Mixture (CT) (ref. 42606)
AESAP1064 or SMASHER™ XL ref. AESAP1100)  SMAC CT Agar (ref. 43391)
®
 Blender bag with filter  SLIDEX E. coli O157 (ref. 417407)
®
 VIDAS ICE kit (bioMérieux ref. 30526, 30 tests) For other specific materials and disposables, please refer
®
 chromID EHEC Agar (EHEC) (bioMerieux ref. 413093 to the Instrument User Manual.
and 413697)
 Cefsulodin WARNINGS AND PRECAUTIONS
 Cefixime
 Vancomycin or Vancomycin Supplement (VANCO  For professional use only.
SUPP) (bioMérieux ref. 412498)  Place the instrument in a room designed for
 Acriflavine microbiological analysis.
 Comply with Good Laboratory Practice (e.g., standard
EN ISO 7218) (7).
 Pathogenic enterohemorrhagic Escherichia coli
O157 are category 3 T* organisms. Samples should
be handled observing the usual safety precautions
and in accordance with any applicable regulations.

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 The kit contains products of animal origin. Certified Method certified NF VALIDATION (BIO-12/25-05/09)
knowledge of the origin and/or sanitary state of the
Procedure for raw beef and veal (including seasoned
animals does not totally guarantee the absence of
meats) limited to a 25 g test sample
transmissible pathogenic agents. It is therefore
recommended that these products be treated as  In a blender bag with filter, aseptically place:
potentially infectious, and handled observing the usual - X g of sample.
safety precautions (do not ingest; do not inhale). - 9X mL of Buffered Peptone Water preheated at
®
 Do not use the SPR s if the pouch is pierced or if the 41.5 ± 1°C.
®
dot sealing a SPR has come unstuck.  Mix using a paddle blender.
 Do not use visibly deteriorated STRs (damaged foil or  Incubate for 7-24 hours at 41.5 ± 1°C.
plastic).  After incubation, mix the contents of the blender bag
 Do not use reagents after the expiration date indicated manually.
®
on the kit label. If the VIDAS Heat and Go is used, transfer 0.5 mL of
 Do not mix reagents (or disposables) from different lots. enrichment broth into the sample well on the strip. Heat
®
 Kit reagents contain sodium azide which can react with for 5  1 minutes (see the VIDAS Heat and Go User
lead or copper plumbing to form explosive metal azides. Manual). Remove the strip and leave to cool for at least
If any liquid containing sodium azide is disposed of in 10 minutes.
the plumbing system, drains should be flushed with If a water-bath is used, transfer 1-2 mL of enrichment
water to avoid build-up. broth into a tube. Seal the tube. Heat for 5  1 minutes
 The substrate in well 10 contains an irritant agent (6.6% at 95-100°C. Cool the tube (do not mix). Transfer 0.5 mL
®
diethanolamine). Refer to the hazard statements "H" of boiled broth into the sample well on the VIDAS strip.
and the precautionary statements "P" above. Note: For samples which coagulate after heating, it is
 Spills should be wiped up thoroughly after treatment recommended to mix the boiled broth using a vortex-
with liquid detergent or a solution of household bleach type mixer.
®
containing at least 0.5 % sodium hypochlorite. See the  Perform the VIDAS assay.
User Manual for cleaning spills on or in the instrument. Note: The non-heated enrichment broth can be stored
®
Do not autoclave solutions containing bleach. for 48 hours at 2-8°C before the VIDAS test is
 The instrument should be regularly cleaned and performed.
decontaminated (see the User Manual).  Confirm the positive results.
Note: If confirmation is not initiated immediately after a
®
STORAGE CONDITIONS positive VIDAS test, store the enrichment broth at
® 2-8°C. Confirmation must be initiated within 48 hours
 Store the VIDAS ECPT kit at 2-8°C. following the end of incubation.
 Do not freeze reagents.
 Store all unused reagents at 2-8°C. Procedure for raw beef and veal limited to a 25 g test
®
 After opening the kit, check that the SPR pouch is sample (including seasoned meats) (protocol
®
correctly sealed and undamaged. If not, do not use the harmonized with VIDAS SPT)
®
SPR s.  In a blender bag with filter, aseptically place:
 Carefully reseal the pouch with the desiccant inside - X g of sample.
®
after use to maintain stability of the SPR s and - 9X mL of Buffered Peptone Water preheated at
return the complete kit to 2-8°C. 41.5 ± 1°C + Y mL (± 5%) of Vancomycin Supplement
 If stored according to the recommended conditions, all (refer to the package insert for Vancomycin
components are stable until the expiration date indicated Supplement ref. 412498) or 8 mg/L of Vancomycin.
on the label. Note 1: If the 1/10 dilution in Buffered Peptone Water
is also used for the enumeration of quality indicator
SAMPLES (PREPARATION) organisms, follow the recommendations in the
The following protocols are recommended. standard ISO 7218 (7). Take this sampling portion into
Frozen samples must be thawed beforehand. account when initially weighing the sample. Sampling
Incubation conditions may have repercussions on short should be performed before adding the supplement or
detection procedures. The temperatures indicated must the Vancomycin.
Note 2: The supplement or the Vancomycin can be
be scrupulously respected.
added directly to the Buffered Peptone Water if the
In particular, it is advisable to ensure that the conditions
enumeration of quality indicator organisms is not
for preheating the enrichment broth enable the indicated
performed using the same test sample.
temperature to be reached. The sample preparation time
 Mix using a paddle blender.
(time between the end of the enrichment broth pre-heating
 Incubate for 16-24 hours at 41.5 ± 1°C.
phase and the start of the food sample incubation phase),
must not exceed 45 minutes. It is recommended to use a
ventilated incubator for the incubation phase.

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VIDAS UP E. coli O157 (including H7) (ECPT) 14080 O - en - 2018/02

 After incubation, mix the contents of the blender bag  After incubation, mix the contents of the blender bag
manually. manually.
® ®
If the VIDAS Heat and Go is used, transfer 0.5 mL of If the VIDAS Heat and Go is used, transfer 0.5 mL of
enrichment broth into the sample well on the strip. Heat enrichment broth into the sample well on the strip. Heat
® ®
for 5  1 minutes (see the VIDAS Heat and Go User for 5  1 minutes (see the VIDAS Heat and Go User
Manual). Remove the strip and leave to cool for at least Manual). Remove the strip and leave to cool for at least
10 minutes. 10 minutes.
®
If a water-bath is used, transfer 2-3 ml of the enrichment Note: Do not use the VIDAS Heat and Go for poultry
broth into a tube (for samples showing strong samples.
coagulation after heating, it is recommended to heat a If a water-bath is used, transfer 1-2 mL of enrichment
larger volume (example: 10 mL)). Seal the tube. Heat for broth into a tube. Seal the tube. Heat for 5  1 minutes
5  1 minutes at 95-100°C. Cool the tube. Mix the boiled at 95-100°C. Cool the tube. Mix the boiled broth using a
broth using a vortex-type mixer and transfer 0.5 mL into vortex-type mixer and transfer 0.5 mL into the sample
® ®
the sample well on the VIDAS strip. well on the VIDAS strip.
® ®
 Perform the VIDAS assay.  Perform the VIDAS assay.
Note: The non-heated enrichment broth can be stored Note: The non-heated enrichment broth can be stored
® ®
for 72 hours at 2-8°C before the VIDAS test is for 48 hours at 2-8°C before the VIDAS test is
performed. performed.
 Confirm the positive results.  Confirm the positive results.
Note: If confirmation is not initiated immediately after a Note: If confirmation is not initiated immediately after a
® ®
positive VIDAS test, store the enrichment broth at positive VIDAS test, store the enrichment broth at
2-8°C. Confirmation must be initiated within 72 hours 2-8°C. Confirmation must be initiated within 48 hours
following the end of incubation. following the end of incubation.
Procedure for raw beef and veal for a test sample Procedure for raw vegetables
between 50 and 375 g (excluding seasoned meats)  In a blender bag with filter, aseptically place:
®
(protocol harmonized with VIDAS SPT) - X g (or X mL) of sample.
 In a blender bag with filter, aseptically place: Note: In the context of NF VALIDATION mark, no test
- X g of sample. samples over 25 g were tested.
- 3X mL of Buffered Peptone Water preheated at - 9X mL of Buffered Peptone Water preheated at
41.5 ± 1°C + Y mL (± 5%) of Vancomycin Supplement 41.5 ± 1°C + Y mL (± 5%) of Vancomycin Supplement
(refer to the package insert for Vancomycin (refer to the package insert for Vancomycin
Supplement ref. 412498) or 8 mg/L of Vancomycin. Supplement ref. 412498) or 8 mg/L of Vancomycin.
 Mix using a paddle blender.  Mix using a paddle blender.
 Incubate for 8-24 hours at 41.5 ± 1°C.  Incubate for 8-24 hours at 41.5 ± 1°C.
 After incubation, mix the contents of the blender bag  After incubation, mix the contents of the blender bag
manually. manually.
®
Transfer 2-3 mL of the enrichment broth into a tube. If the VIDAS Heat and Go is used, transfer 0.5 mL of
Seal the tube. Heat in a water-bath for 5  1 minutes at enrichment broth into the sample well on the strip. Heat
®
95-100°C. Cool the tube (do not mix). Transfer 0.5 mL of for 5  1 minutes (see the VIDAS Heat and Go User
®
boiled broth into the sample well on the VIDAS strip. Manual). Remove the strip and leave to cool for at least
Note: For samples which coagulate after heating, or for 10 minutes.
®
a protocol that is harmonized with the VIDAS UP If a water-bath is used, transfer 1-2 mL of enrichment
Salmonella method, it is recommended to mix the boiled broth into a tube. Seal the tube. Heat for 5  1 minutes
broth using a vortex-type mixer. at 95-100°C. Cool the tube. Mix the boiled broth using a
®
 Perform the VIDAS assay. vortex-type mixer and transfer 0.5 mL into the sample
®
Note: The non-heated enrichment broth can be stored well on the VIDAS strip.
® ®
for 48 hours at 2-8°C before the VIDAS test is  Perform the VIDAS assay.
performed. Note: The non-heated enrichment broth can be stored
®
 Confirm the positive results. for 48 hours at 2-8°C before the VIDAS test is
Note: If confirmation is not initiated immediately after a performed.
®
positive VIDAS test, store the enrichment broth at  Confirm the positive results.
2-8°C. Confirmation must be initiated within 48 hours Note: If confirmation is not initiated immediately after a
®
following the end of incubation. positive VIDAS test, store the enrichment broth at
Note: Prolonging the incubation period up to 24 hours 2-8°C. Confirmation must be initiated within 48 hours
improves the performance of the alternative method. following the end of incubation.
Note: Prolonging the incubation period up to 24 hours
Procedure for all raw meat products
improves the performance of the alternative method.
 In a blender bag with filter, aseptically place:
- X g (or X mL) of sample.
Note: In the context of NF VALIDATION mark, no test
samples over 25 g were tested.
- 9X mL of Buffered Peptone Water preheated at
41.5 ± 1°C + Y mL (± 5%) of Vancomycin Supplement
(refer to the package insert for Vancomycin
Supplement ref. 412498) or 8 mg/L of Vancomycin.
 Mix using a paddle blender.
 Incubate for 16-24 hours at 41.5 ± 1°C.

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Note: If confirmation is not initiated immediately after a

Procedure for raw milk and raw milk dairy products ®
positive VIDAS test, store the enrichment broth at
 In a blender bag with filter, aseptically place: 2-8°C. Confirmation must be initiated within 48 hours
- X g (or X mL) of sample. following the end of incubation
Note 1: In the context of NF VALIDATION mark, no
test samples over 25 g were tested. Protocol outside the scope of NF VALIDATION
- 9X mL of Buffered Peptone Water at 18-25°C (7) + Procedure for animal feed products, soil and breeding
Acriflavine (10 mg/L). environmental samples
 Mix using a paddle blender.
 Incubate for 20-26 hours at 41.5 ± 1°C.  In a blender bag with filter, aseptically place:
 After incubation, mix the contents of the blender bag - X g (or X mL) of sample.
manually. - 9X mL of Buffered Peptone Water preheated at
®
If the VIDAS Heat and Go is used, transfer 0.5 mL of 41.5 ± 1°C + Vancomycin (8 mg/L) + Cefixime
(0.0125 mg/L) + Cefsulodin (10 mg/L) (VCC).
enrichment broth into the sample well on the strip. Heat
®  Mix using a paddle blender.
for 5  1 minutes (see the VIDAS Heat and Go User
 Incubate for 16-24 hours at 41.5 ± 1°C.
Manual). Remove the strip and leave to cool for at least
 After incubation, mix the contents of the blender bag
10 minutes.
manually.
If a water-bath is used, transfer 1-2 mL of enrichment ®
If the VIDAS Heat and Go is used, transfer 0.5 mL of
broth into a tube. Seal the tube. Heat for 5  1 minutes
enrichment broth into the sample well on the strip. Heat
at 95-100°C. Cool the tube. Mix the boiled broth using a ®
for 5  1 minutes (see the VIDAS Heat and Go User
vortex-type mixer and transfer 0.5 mL into the sample
® Manual). Remove the strip and leave to cool for at least
well on the VIDAS strip.
® 10 minutes.
 Perform the VIDAS assay.
If a water-bath is used, transfer 1-2 mL of enrichment
Note: The non-heated enrichment broth can be stored
® broth into a tube. Seal the tube. Heat for 5  1 minutes
for 72 hours at 2-8°C before the VIDAS test is
at 95-100°C. Cool the tube. Mix the boiled broth using a
performed.
vortex-type mixer and transfer 0.5 mL into the sample
 Confirm the positive results. ®
well on the VIDAS strip.
Note: If confirmation is not initiated immediately after a ®
®  Perform the VIDAS assay.
positive VIDAS test, store the enrichment broth at
Note: The non-heated enrichment broth can be stored
2-8°C. Confirmation must be initiated within 72 hours ®
for 48 hours at 2-8°C before the VIDAS test is
following the end of incubation.
performed.
Procedure for production environmental samples  Confirm the positive results.
 In a blender bag with filter or in a suitable container, Note: If confirmation is not initiated immediately after a
®
aseptically place: positive VIDAS test, store the enrichment broth at
- X g (or X mL) of sample. 2-8°C. Confirmation must be initiated within 48 hours
Note 1: For environmental surface samples, the following the end of incubation.
collection device should first be dampened with a Confirmation of positive results obtained using the
sterile diluent (e.g. Buffered Peptone Water) method certified NF VALIDATION and the procedures
containing, if necessary, a suitable neutralizing agent outside the scope of NF VALIDATION
(e.g. Lecithin-Polysorbate-L.Histidine- Sodium
Thiosulfate mixture). After collection, place the device In the context of NF VALIDATION mark, all positive
®
in a suitable volume of enrichment broth (e.g.: swab in results obtained with VIDAS ECPT must be confirmed.
10 mL, sampling pad in 100 mL). For the 7 or 8-hour enrichment procedures, it is
Note 2: In the context of NF VALIDATION mark, no recommended to prolong incubation of the broth for a total
test samples over 25 g were tested. duration of 20-24 hours.
- 9X mL of Buffered Peptone Water preheated at
41.5 ± 1°C + Vancomycin (8 mg/L) + Cefixime Follow one of the 3 validated procedures below:
(0.0125 mg/L) + Cefsulodin (10 mg/L) (VCC). 1. Isolate the non-heated enrichment broth onto SMAC
 Mix using a paddle blender. CT Agar and chromID® EHEC Agar with Cefixime and
 Incubate for 15-24 hours at 41.5 ± 1°C. Tellurite (CT-EHEC) and incubate the plates according
 After incubation, mix the contents of the blender bag to the recommendations in the package inserts.
manually. Identify between one and five typical colonies using
®
If the VIDAS Heat and Go is used, transfer 0.5 mL of one of the 3 options for identification described below.
®
enrichment broth into the sample well on the strip. Heat 2. Perform immuno-concentration with VIDAS ICE
®
®
for 5  1 minutes (see the VIDAS Heat and Go User followed by isolation onto chromID EHEC (without the
Manual). Remove the strip and leave to cool for at least Cefixime – Tellurite mixture) and onto SMAC CT Agar
10 minutes. and incubate the plates according to the
If a water-bath is used, transfer 1-2 mL of enrichment recommendations in the package inserts.
broth into a tube. Seal the tube. Heat for 5  1 minutes Identify between one and five typical colonies using
at 95-100°C. Cool the tube. Mix the boiled broth using a one of the 3 options for identification described below.
vortex-type mixer and transfer 0.5 mL into the sample 3. Perform a method validated according to the standard
®
well on the VIDAS strip. EN ISO 16140 based on a different principle. The
®
 Perform the VIDAS assay. validated protocol of the second method should be
Note: The non-heated enrichment broth can be stored followed entirely (e.g. enrichment duration).
®
for 48 hours at 2-8°C before the VIDAS test is
performed.
 Confirm the positive results.

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 Perform the VIDAS® assay.

Options for confirmation:
Store the remaining non-heated broth at 2-8°C in case it
 Identify between one and five typical colonies using the
is required for confirmation purposes.
conventional tests described in the methods
 Confirm the positive results.
standardized by the CEN or ISO (including the
purification step) (8). Procedure for irrigation water samples
 A bioMérieux strip and a latex O157 test can be used to  In a blender bag with filter, aseptically place:
directly test isolated colonies (without the purification - 25 g of sample.
step). - 225 mL of Buffered Peptone Water preheated at
®
 Using the chromID EHEC Agar, perform a latex O157 41.5 ± 1°C + Vancomycin (8 mg/L) + Cefixime
test directly on isolated colonies. (0.0125 mg/L) + Cefsulodin (10 mg/L) (VCC).
In the event of discordant results (positive with the  Mix for 2 minutes using a paddle blender.
alternative method, not confirmed by one of the options  Incubate for 8-24 hours at 41.5 ± 1°C.
described above, and particularly the latex test), the  After incubation, mix the contents of the blender bag
laboratory must take the necessary steps to ensure that manually.
®
the results obtained are accurate. If the VIDAS Heat and Go is used, transfer 0.5 mL of
For instance, isolation may be performed again using the enrichment broth into the sample well on the strip. Heat
®
procedure (1 or 2) that was not followed the first time. for 5  1 minutes (see the VIDAS Heat and Go User
Manual). Remove the strip and leave to cool for at least
AOAC RI approved protocols (N°060903) 10 minutes.
If a water-bath is used, transfer 1-2 mL of enrichment
Procedure for raw beef and beef trim limited to a 25 g
broth into a tube. Seal the tube. Heat for 5  1 minutes
test sample
at 95-100°C. Cool the tube. Mix the boiled broth using a
 In a blender bag with filter, aseptically place: vortex-type mixer and transfer 0.5 mL into the sample
- 25 g of sample. ®
well on the VIDAS strip.
- 225 mL of Buffered Peptone Water preheated at ®
 Perform the VIDAS assay.
41.5 ± 1°C. Store the remaining non-heated enrichment broth at
 Mix for 2 minutes using a paddle blender. 2-8°C in case it is required for confirmation purposes.
 Incubate for 7-24 hours at 41.5 ± 1°C.  Confirm the positive results.
 After incubation, mix the contents of the blender bag
manually. Procedure for raw vegetables
®
If the VIDAS Heat and Go is used, transfer 0.5 mL of  In a blender bag with filter, aseptically place:
enrichment broth into the sample well on the strip. Heat - 25 g of sample.
®
for 5  1 minutes (see the VIDAS Heat and Go User - 225 mL of Buffered Peptone Water preheated at
Manual). Remove the strip and leave to cool for at least 41.5 ± 1°C + 1 mL of Vancomycin Supplement (refer
10 minutes. to the package insert for Vancomycin Supplement Ref.
If a water-bath is used, transfer 1-2 mL of enrichment 412498) or 8 mg/L of Vancomycin.
broth into a tube. Seal the tube. Heat for 5  1 minutes  Mix for 2 minutes using a paddle blender.
at 95-100°C. Cool the tube (do not mix). Transfer  Incubate for 8-24 hours at 41.5 ± 1°C.
0.5 mL of boiled broth into the sample well on the  After incubation, mix the contents of the blender bag
®
VIDAS strip. manually.
®
 Perform the VIDAS assay. ®
If the VIDAS Heat and Go is used, transfer 0.5 mL of
Store the remaining non-heated broth at 2-8°C in case it enrichment broth into the sample well on the strip. Heat
®
is required for confirmation purposes. for 5  1 minutes (see the VIDAS Heat and Go User
 Confirm the positive results. Manual). Remove the strip and leave to cool for at least
10 minutes.
Procedure for raw beef and beef trim for a 75 g or
If a water-bath is used, transfer 1-2 mL of enrichment
375 g test sample
broth into a tube. Seal the tube. Heat for 5  1 minutes
 Aseptically place 75 g or 375 g of sample in a blender at 95-100°C. Cool the tube. Mix the boiled broth using a
bag with filter. vortex-type mixer and transfer 0.5 mL into the sample
Note: Frozen samples must be thawed beforehand. ®
well on the VIDAS strip.
 Add 225 mL or 1125 mL respectively of Buffered ®
 Perform the VIDAS assay.
Peptone Water preheated at 41.5 ± 1°C + Y mL (± 5%) Store the remaining non-heated enrichment broth at
of Vancomycin Supplement (refer to the package insert 2-8°C in case it is required for confirmation purposes.
for Vancomycin Supplement Ref. 412498) or 8 mg/L of  Confirm the positive results.
Vancomycin.
 Mix for 2 minutes using a paddle blender.
 Incubate for 8-24 hours at 41.5 ± 1°C.
Note: For 375 g raw beef samples, incubate for
10-24 hours.
 After incubation, mix the contents of the blender bag
manually.
Transfer 2-3 mL of the enrichment broth into a tube.
Seal the tube. Heat in a water-bath for 5  1 minutes at
95-100°C. Cool the tube (do not mix). Transfer 0.5 mL
®
of boiled broth into the sample well on the VIDAS strip.

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VIDAS UP E. coli O157 (including H7) (ECPT) 14080 O - en - 2018/02

Confirmation of positive results obtained using the Procedure

AOAC RI approved protocols 1. Only remove the required reagents from the
All positive results obtained with VIDAS® ECPT must be refrigerator and allow them to come to room
confirmed. temperature for 30 minutes before use.
Confirmation should be performed using the non-heated 2. Use one “ECPT” strip and one “ECPT” SPR® for each
enrichment broth stored at 2-8°C. sample, control or standard to be tested. Make sure
Follow one of the 2 validated procedures below: the storage pouch has been carefully resealed
®
®
1. Isolate onto SMAC CT Agar or chromID EHEC Agar after the required SPR s have been removed.
with Cefixime and Tellurite (CT-EHEC) and incubate 3. The test is identified by the "ECPT" code on the
the plate according to the recommendations in the instrument. The standard must be identified by "S1"
package insert. and tested in duplicate.
2. Perform immuno-concentration with VIDAS® ICE If the positive control needs to be tested, it should be
followed by isolation onto chromID® EHEC Agar identified by "C1".
(without the Cefixime – Tellurite mixture) or onto If the negative control needs to be tested, it should be
SMAC CT Agar and incubate the plate for according to identified by "C2".
the recommendations in the package insert. 4. If necessary, mix the standard and controls using a
If there is a discrepancy between a positive VIDAS
® vortex-type mixer and then dispense 500 µL into the
ECPT test and the absence of typical colonies on sample well.
®
chromID EHEC Agar, the colonies must be isolated on Note: Do not heat the standard and controls.
SMAC CT Agar. 5. For the heating step and for the transfer of the sample
Typical colonies are confirmed using the USDA method into the strip, refer to the procedure used.
(9) or the FDA BAM Reference Method (10). ®
6. Insert the SPR s and the strips into the instrument.
Check to make sure the color labels with the assay
®
INSTRUCTIONS FOR USE code on the SPR s and the Reagent Strips match.
For complete instructions, see the User Manual. 7. Initiate the assay as directed in the User Manual. All
® the assay steps are performed automatically by the
Reading VIDAS PTC protocol data and MLE data instrument. The results are obtained within
When using the assay for the first time approximately 50 minutes.
With the external instrument bar code reader, 8. After the assay is completed, remove the SPR®s and
1. Scan the PTC bar code(s) downloadable from strips from the instrument.
www.biomerieux.com/techlib. 9. Dispose of the used SPR®s and strips into an
This reading allows VIDAS® PTC protocol data to be appropriate biohazard receptacle in accordance with
transferred to the instrument software for its update. applicable local regulations.
2. read the MLE data
Note: If the MLE data have been read before the RESULTS AND INTERPRETATION
®
VIDAS PTC protocol, read the MLE data again. Once the assay is completed, results are analyzed
automatically by the computer.
When opening a new lot of reagents Fluorescence is measured twice in the Reagent Strip’s
Enter the specifications (or factory master data) into the reading cuvette for each sample tested.
instrument using the master lot entry (MLE) data. If this The first reading is a background reading of the substrate
®
operation is not performed before initiating the tests, the cuvette before the SPR is introduced into the substrate.
instrument will not be able to print results. The second reading is taken after incubating the substrate
®
It is possible to enter the MLE data manually or with the enzyme remaining on the interior of the SPR .
automatically depending on the instrument (refer to the
User Manual). The RFV (Relative Fluorescence Value) is calculated by
Note: the master lot data need only be entered once subtracting the background reading from the final result.
for each lot. This calculation appears on the result sheet.
The RFV obtained for each sample is interpreted by the
Calibration instrument as follows:
Calibration, using the standard provided in the kit, must be sample RFV
Test value =
performed each time a new lot of reagents is opened, standard RFV
after the master lot data have been entered. Calibration
should then be performed every 14 days. This operation Thresholds and interpretations
provides instrument-specific calibration curves and Test value Interpretation
compensates for possible minor variations in assay signal
throughout the shelf-life of the kit. < 0.04 Negative
The standard, identified by S1, must be tested in  0.04 Positive
duplicate (see the User Manual). The standard value
must be within the set RFV "Relative Fluorescence Value" The printed report includes:
range. If this is not the case, recalibrate.  the type of test performed,
 the sample identification,
 the date and time,
 the lot number and expiration date of the kit,
 the RFV, Test Value and interpreted result for each
sample.

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VIDAS UP E. coli O157 (including H7) (ECPT) 14080 O - en - 2018/02

A result with a test value that is less than the threshold Caution
value indicates that the sample does not contain E. coli ®
The VIDAS ECPT assay has been evaluated on a large
O157 or contains E. coli O157 at a concentration below number of products. Given the wide variety of products
the detection limit. and manufacturing procedures, it is recommended to
A result with a test value that is greater than or equal to check that the composition of the matrices tested does not
®
the threshold value indicates a sample contaminated with affect the reliability of the VIDAS ECPT results.
E. coli O157. In this case, refer to the section on The VIDAS® Heat and Go system has been evaluated on
"Confirmation of positive results". a large number of food matrices. Given the wide variety of
Invalid results are reported: food matrices and manufacturing procedures, when
 when the background reading is above a pre- commissioning the system, it is recommended to verify
determined cut-off (indicating substrate contamination). that the heating step does not result in a substantial
In this case, repeat the assay with the heated broth or coagulation or precipitation of the sample in the sample
the reagent concerned (S1, C1 or C2). well as this could lead to an incorrect volume of sample
®
 if there is no standard available for the lot number of the being taken into the SPR .
®
sample test strip. Note: Do not use the VIDAS Heat and Go for egg
In this case, run a standard in duplicate in strips with the products and poultry samples.
same lot number as the invalid sample test. The sample
test result can then be recalculated using the new stored PERFORMANCE
standards. See the User Manual for complete The performance data obtained in the context of NF
information. VALIDATION certification are available in the synthesis
report on the AFNOR certification website: http://nf-
QUALITY CONTROL validation.afnor.org/en.
One positive control and one negative control are included
® All of the results were obtained using bioMerieux Buffered
in each VIDAS ECPT kit.
Peptone Water.
These controls must be performed each time a new lot of
reagents is opened to verify the calibration (see section ®
The VIDAS UP E. coli O157 (including H7) (ECPT)
“Calibration”). method has been certified NF VALIDATION as an
It is also recommended to perform the controls each time alternative analysis method for the detection of E. coli
new kits are received to ensure that reagent performance O157 in raw meat products, raw vegetables, raw milk
has not been altered. dairy products and production environmental
The instrument will only be able to check the control samples.
values if they are identified by C1 and C2. This validation has been obtained by comparison with
Results cannot be validated if the control values deviate the reference method described in the international
from the expected values. standard EN ISO 16654 (8) according to the standard
Note EN ISO 16140-2 (12).
The BIO-12/25-05/09 validation certificate can be
It is the responsibility of the user to perform Quality obtained from our Technical Assistance Service or
Control in accordance with any applicable local from AFNOR Certification. The date of end of validity
regulations. for the NF VALIDATION certification is indicated on
the certificate.
LIMITATIONS OF THE METHOD
®
The VIDAS ECPT assay alerts the user to the possible
presence of E. coli O157 including H7. The user is
therefore more vigilant during the confirmation process.
BIO-12/25-05/09
Reactions have been observed with strains, such as ALTERNATIVE ANALYTICAL METHODS FOR AGRIBUSINESS
group N Salmonella, that possess the same surface Certified by AFNOR Certification
http://nf-validation.afnor.org/en
receptors as E. coli O157 including H7 (11). These
strains, which are non-typical on selective agar used for
confirmation, can be at the origin of rare discrepancies The VIDAS® UP E. coli O157 (including H7) (ECPT)
®
between a VIDAS ECPT test result and the result of the method has been validated and certified by the AOAC
confirmation test. Research Institute as a Performance Tested Method
Other rare discrepancies can be obtained with sorbitol- (Certificate No. 060903) for the detection of E. coli
positive strains of E. coli O157 including H7 which are O157 in a variety of foods.
®
positive with the VIDAS ECPT test and non-typical on
®
the confirmation agar. The principle of the chromID
EHEC agar is based on the dual sorbitol-negative and PERFORMANCE TESTED METHOD
 glucuronidase-negative characteristic of E. coli O157:H7 Certified by AOAC Research Institute
www.aoac.org
or O157:H- strains. The non-H7 E. coli strains ( The following matrices were included in the AOAC
glucuronidase-positive) are positive with the validation: raw beef, beef trim, irrigation water, fresh
®
VIDAS ECPT test and non-typical on this agar. This can spinach, bagged lettuce.
also be the case for the rare strains of E. coli O157:H7
that are sorbitol-negative but  glucuronidase-positive.

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VIDAS UP E. coli O157 (including H7) (ECPT) 14080 O - en - 2018/02

WASTE DISPOSAL INDEX OF SYMBOLS

Dispose of used or unused reagents as well as any other Symbol Meaning
contaminated disposable materials following procedures
for infectious or potentially infectious products. Catalog number
It is the responsibility of each laboratory to handle waste
and effluents produced according to their nature and Manufacturer
degree of hazardousness and to treat and dispose of
them (or have them treated and disposed of) in Temperature limit
accordance with any applicable regulations.
Use by date
LITERATURE REFERENCES
1. HOCKIN J., LIOR H. - Hemorrhagic colitis and haemolytic Batch code
uremic syndrome caused by Escherichia coli O157:H7 in
Canada - Can. Dis. Weekly Rep. - Health Welfare Can. - Consult Instructions for Use
1987, vol. 13, p. 203-204.
2. PUDDEN D., TUTTLE N., KORN D., et al. - Hemorrhagic
colitis in a nursing home-Ontario - Can. Dis. Weekly Rep. - Contains sufficient for <n> tests
1985, vol. 11, p. 169-170.
3. KNIGHT P. - Hemorrhagic E. coli : the danger increases. Date of manufacture
ASM news - 1993, vol. 59, n° 5, p. 247 - 250.
4. FIELDS P.I., BLOM K., HUGUES H.J., et al - Molecular
characterization of the gene encoding H antigen in LIMITED WARRANTY
Escherichia coli and development of a PCR-restriction
bioMérieux warrants the performance of the product for its
fragment length polymorphism test for identification of E. coli
O157:H7 and O157:NM - J. Clinical microbiol. - 1997, vol. 35, stated intended use provided that all procedures for
n° 5, p. 1066-1070. usage, storage and handling, shelf life (when applicable),
5. CHAHED A., GHAFIR Y., DAUBE G., et al. – Survey of the and precautions are strictly followed as detailed in the
contamination of foodstuffs of animal origin by Shiga toxin instructions for use (IFU).
producing Escherichia coli serotype O157:H7 in Belgium from Except as expressly set forth above, bioMérieux hereby
1999 to 2003 – Eurosurveillance – 2005, vol. 10, n°3, disclaims all warranties, including any implied warranties
p. 33-36. of merchantability and fitness for a particular purpose or
6. AL-SAIGH H., ZWEIFEL C., BLANCO J. et al – Fecal
shedding of Escherichia coli O157, Salmonella, and use, and disclaims all liability, whether direct, indirect or
Campylobacter in Swiss cattle at slaughter – Journal of food consequential, for any use of the reagent, software,
®
protection - 1 April 2004, vol. 67, n°4, p. 679 – 684 instrument and disposables (the “System”) other than as
7. Microbiology of food and animal feeding stuffs - General set forth in the IFU.
requirements and guidance for microbiological examinations
– EN ISO 7218
8. Microbiology of food and animal feeding stuffs – Horizontal
method for the detection of Escherichia coli O157 - EN ISO
16654
9. De BOER E. and HEUVELINK A.E. – methods for the
detection and isolation of Shiga toxin-producing Escherichia
coli - Journal of applied microbiology symposium – 2000, vol.
88, p. 133S-143S.
10. Microbiology of food and animal feeding stuffs – Protocol for
the validation of alternative methods - EN ISO 16140 - 2003.
11. U.S. Department of Agriculture/Food Safety Inspection
Services - Microbiological Laboratory Guidelines -
http://www.fsis.usda.gov/Science/Microbiological_Lab_Guide
book/index.asp.
12. Microbiology of the food chain – Method validation – Part 2:
Protocol for the validation of alternative (proprietary) methods
against a reference method – EN ISO 16140-2

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VIDAS UP E. coli O157 (including H7) (ECPT) 14080 O - en - 2018/02

REVISION HISTORY
Change type categories:
N/A Not applicable (First publication)
Correction Correction of documentation anomalies
Technical change Addition, revision and/or removal of information related to the product
Administrative Implementation of non-technical changes noticeable to the user
Note: Minor typographical, grammar, and formatting changes are not included in the revision history.

Release date Part Number Change Type Change Summary

Index of symbols
Administrative
Creation of the revision history table
2015/01 14080N
Content of the kit
Technical change
Warnings and precautions
Limitations of the method
Administrative
Limited warranty
Reagents, materials and disposables required but not
2018/02 14080O provided
Technical change Samples (preparation)
Performance
Literature references

BIOMERIEUX, the BIOMERIEUX logo, CHROMID, SLIDEX, SMASHER, SPR and VIDAS are used, pending, and/or registered trademarks belonging to
bioMérieux, or one of its subsidiaries, or one of its companies.
Any other name or trademark is the property of its respective owner.

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