GOLD STANDARD DIAGNOSTICS E Coli O145 IMS Beads Instructions
- September 25, 2024
- GOLD STANDARD DIAGNOSTICS
Table of Contents
E Coli O145 IMS Beads
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Product Information
Specifications:
Product Name: E. coli O145 IMS Beads
Description: Immunomagnetic Separation Beads
for E. coli O145
Product No.: 543050 (2 mL)
Product Usage Instructions
Safety Instructions
Biological Waste: Decontaminate biological
waste by autoclaving or using another effective method. Dispose of
samples following local, state, and federal regulations.
Limitations of the Test
If a positive result is obtained on an unknown organism, further
testing such as PCR should be carried out for confirmation.
Exercise good judgment when interpreting test results, especially
with preliminary positive results.
E. Coli O145 Detection Scheme in Food Samples
Screening Using Real-Time PCR
Enriched samples are screened for the presence of stx and eae
genes. Samples with positive results on the initial stx/eae PCR
screen will undergo three additional Real-time PCR assays to
determine the presence of one of the top six serogroups (O26, O45,
O103, O111, O121, or O145).
Confirmation
Latex-positive colonies are confirmed by PCR assays followed by
biochemical identification.
Materials Provided
-
Superparamagnetic beads attached with purified antibodies
against E. coli O145 covalently bound to the surface. The beads are
supplied in a suspension of PBS, pH 7.4 with 0.05% Sodium Azide, 2
mL vial. -
User’s guide.
Additional Materials Required for Usage
-
Sterile water for reagent preparation.
-
Sterile PBS buffer: Dissolve 8g NaCl, 0.2g KCl, 1.44g Na2HPO4,
0.24g KH2PO4 in 800mL distilled water, adjust pH to 7.4 with HCl,
and bring volume to 1L with distilled water. -
Sterile Tryptic Soy Broth (TSB).
-
Sterile Tryptic Soy Agar (TSA).
-
Ethanol (70% v/v).
FAQ
Q: What should I do if I encounter a positive result on
an unknown organism?
A: If a positive result is obtained on an
unknown organism, further testing such as PCR should be carried out
for confirmation.
Q: How should I dispose of biological
waste?
A: Biological waste should be decontaminated by
autoclaving or using another effective method. Dispose of samples
following local, state, and federal regulations.
Q: What materials are provided with the E. coli O145 IMS
Beads?
A: The product includes superparamagnetic beads
attached with purified antibodies against E. coli O145 covalently
bound to the surface and a user’s guide.
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Importance of STEC Determination Shiga toxin-producing Escherichia coli
strains (non-O157 STEC) have become an increasing public health concern. Some
of the non-O157 STEC possess the same range of virulence factors as E. coli
O157:H7, including the locus of enterocyte effacement and production of Shiga
toxin. STEC has been implicated in numerous outbreaks, causing serious illness
(hemolytic uremic syndrome), or death.
A study from the CDC showed that from 1982 to 2002 approximately 70% of
non-O157 STEC infections in the USA were caused by strains from one of six
major serogroups: O26, O45, O103, O111, O121, and O145. Non O-157 STEC has
been found in ground beef and in cattle hides, and in feces at levels
comparable to those of E. coli O157. Bovine feces can be a source of
environmental contamination (soil, water) which can lead to secondary
contamination of produce growing in fields.
It is difficult to distinguish pathogenic non-O157 STEC strains from non-
pathogenic E. coli strains because the former rarely possess any
distinguishing phenotypic or biochemical characteristics from the latter.
Therefore, methods such as PCR and latex agglutination tests have been
developed by the USDA-Agricultural Research Service Eastern Regional Research
Center (USDA- ARS-ERRC) to help on the identification of these STEC strains.
In order to conduct the confirmation methods, enrichment of the sample
followed by immunomagnetic (IMS) separation is performed. The IMS method
described in this user’s guide is part of the testing protocol utilized and
mandated by the FSIS for testing ground beef and beef trim, and described in
the USDA-FSIS Microbiology Laboratory Guidebook (MLG) Chapter 5 “Detection,
Isolation and Identification of Top Seven Shiga ToxinProducing Escherichia
coli (STECs) from Meat Products and Carcass and Environmental Sponges”.
General Limited Warranty: Gold Standard Diagnostics warrants the products
manufactured by the Company, against defects and workmanship when used in
accordance with the applicable instructions for a period not to ex tend beyond
the product’s printed expiration date. Gold Standard Diagnostics makes no
other warranty, expressed or implied. There is no warranty of merchantability
or fitness for a particular purpose.
For ordering or technical assistance contact: Gold Standard Diagnostics 795 Horsham Road Horsham, PA 19044 WEB: www.abraxiskits.com
Tel.: 215-357-3911 Fax: 215-357-5232 Ordering: info.abraxis@us.goldstandarddiagnostics.com Technical Support: support.abraxis@us.goldstandarddiagnostics.com
Date this User Guide is effective: 05/15/2024
Version: 01
E. coli O145 IMS Beads
Immunomagnetic Separation Beads for E. coli O145
Product No. 543050 (2 mL)
1. General Description The Gold Standard Diagnostics’ E. coli O145 IMS beads
are immunomagnetic separation beads designed for the rapid and selective
concentration of Escherichia coli serogroup O145 to help with their
confirmation on selective confirmation agars. The Gold Standard Diagnostics’
E. coli O145 IMS beads should be used as part of the USDA-FSIS test protocol
described in the USDA-FSIS Microbiology Laboratory Guidebook (MLG) Chapter 5
“Detection, Isolation and Identification of Top Seven Shiga Toxin-Producing
Escherichia coli (STECs) from Meat Products and Carcass and Environmental
Sponges”.
2. Safety Instructions Biological waste should be decontaminated by
autoclaving or by using another effective method. Discard samples according to
local, state and federal regulations.
3. Storage and Stability The E. coli O145 IMS beads should be stored between
2-8°C (do not freeze) and may be used until the last day of the month as
indicated by the expiration date on the label. All reagents and samples to be
analyzed should be at room temperature before use.
4. Test Principle The IMS beads provided in the kit are coupled to antibodies
against E. coli serotype O145. The Gold Standard Diagnostics’ E. coli O145 IMS
beads should be used as part of the USDA-FSIS test protocol described in the
USDA-FSIS Microbiology Laboratory Guidebook (MLG) Chapter 5 “Detection,
Isolation and Identification of Top Seven Shiga Toxin-Producing Escherichia
coli (STECs) from Meat Products and Carcass and Environmental Sponges”. The
MLG utilizes sample enrichment in modified tryptic soy broth, followed by
multiplexed Real Time-PCR assay s targeting stx1, stx2 and eae genes and the
wxz gene in the O-antigen gene clusters of the six serogroups, and then
immunomagnetic separation (IMS) followed by plating onto Rainbow Agar (mRBA)
(Fratamico et al). Colonies are then tested for specific O antigens using
latex agglutination and positive colonies are purified on Sheep Blood Agar
(SBA) and confirmed using PCR and biochemical identification.
5. Limitations of the Test If a positive result is obtained on an unknown
organism, further test such as PCR should be carried out for confirmation.
Apply good judgment to any test result, particularly when preliminary positive
results are observed.
6. Warning and Precautions -This product is for research purposes only; not
for diagnostic or in vivo use. -Do not freeze reagents. -Do not allow reagents
to become contaminated by using dirty transfer pipettes. -Use reasonable
judgment when interpreting the test results. -Prior to use, ensure that the
product has not expired by verifying that the date of use is prior to the
expiration date on the label. – Avoid cross-contamination of samples by using
a new sample stick for each sample. -Specimens may contain pathogenic
organisms, handle with appropriate precautions. -Ensure that reagent bottle
caps are tight after each use to prevent drying of reagents. -Reagents contain
0.05-0.1% sodium azide as a preservative. Sodium azide may react with lead or
copper plumbing to produce metal azides which might cause explosion. To
prevent azide accumulation in plumbing, flush with copious amounts of water
immediately after disposal. -Dispose according to local regulations.
7. Summary of E. Coli O145 in Food Samples Detection Scheme ((MLG) Chapter 5)
7.1 Enrichment 325 +/- 32.5 gm sample is combined with 975 +/- 19.5 mL of
modified TSB + casamino acids and 8 mg/L novobiocin (mTSB), stomached and
incubated static at 43 +/- 1 °C for 15-22 hours. Positive and negative
controls need to be included.
7.2 Screening Using Real-Time PCR Enriched samples are screened for the
presence of stx and eae genes. Samples with positive results (both gene
targets) on the initial stx/eae PCR screen will be tested by three additional
Real-time PCR assays to determine if a top six serogroup (O26, O45, O103,
O111, O121 or O145) is present.
7.3 Immunomagnetic Separation (IMS) Samples positive by the screening test are
potential positives. Isolation of non-O157 STEC is carried out using
immunomagnetic separation with specific anti-serotype beads (see protocol
below, section D). A post-IMS acid treatment is performed to reduce background
flora on the mRBA plate. Following the 1 hour acid treatment, immunomagnetic
beads with adhering bacteria (O145) are diluted 1:1 and 1:10. Then, 0.1 mL is
spread (plated) onto mRBA. Plates are incubated at 35 +/- 2 °C for 20-24
hours.
7.4 Identification Colonies are picked from RBA plates and tested for
agglutination with antisera specific for the serogroup of interest (at this
stage the target serogroup is known). At least one colony of each
morphological type on each plate is tested using O145 latex agglutination kits
(Gold Standard Diagnostics PN 541050). A minimum of five latex positive
colonies from each plate are streaked onto SBA plates and incubated at 35 +/-
2 °C for 18-24 hours. Colonies are then confirmed by specific latex
agglutination test.
7.5 Confirmation Latex positive colonies are confirmed by PCR assay s followed
by biochemical identification.
A. Materials Provided 1. Superparamagnetic beads attached with purified
antibodies against E. coli O145 covalently bound to the
surface. The beads are supplied in a suspension of PBS, pH 7.4 with 0.05%
Sodium Azide, 2 mL vial. 2. User’s guide.
B. Additional Materials (not provided with the test) 1. Pipettes (10-100 µL)
with disposable filter tips 2. 1 mL dispenser pipette 3. mTSB (available from
many media manufacturers) 4. Stomacher bags with or without mesh 5.
Microcentrifuge tubes (1.5 mL) and 50 mL centrifuge tubes. 6. Sterile test
tubes 7. Sterile glassware 8. Sterile inoculating loops, “hockey sticks” or
spreaders 9. E Buffer: add 0.5 g of BSA, and 50 µL of Tween-20 to 100 mL of
Buffered peptone water (BPW). Mix
well. Filter sterilize, pH 7.2 +/- 0.2. (Sigma catalog # A7906-500G) 10.
Separation Columns and Separation Magnets (MACS, Miltenyi Biotec) or Multi-6
Magnetic Separator (Gold
Standard Diagnostics PN 472260) 11. mRBA Plates 12. HCl, 1 N 13. Tube shaker
or agitator 14. Incubator 15. Vortexer 16. Disinfectant Solution, e.g. Sodium
hypochlorite solution >1.3% w/w.
C. Test Preparation 1. All reagents and samples to be analyzed should be at
room temperature before use. 2. Thoroughly suspend the IMS beads by repeated
gentle inversion of the vial.
D. Assay Procedure (Using Gold Standard Diagnostics Magnetic Separator)
Please refer to the USDA-FSIS Microbiology Laboratory Guidebook (MLG) Chapter
5.
1. For each sample positive for one of the top six serogroups, transfer 2-5
mL from the overnight enrichment through a 40 µm cell strainer into a 50 mL
conical centrifuge tube.
2. Transfer 1.0 mL aliquot of the enrichment filtrate sample to a sterile 1.5
mL centrifuge tube. 3. Add 20 µL of the IMS O26 beads to the sample and vortex
for 10-15 seconds to mix. 4. Incubate the sample(s) at room temperature (18-30
°C) with rocking for 15 minutes. 5. Place the sample tube(s) onto the magnetic
separation rack. Allow to separate for 3 minutes. 6. Using a pipette,
carefully remove the liquid from the sample tube (NOTE: be careful so as not
to
remove or touch the IMS beads on the side of the tube closest to the magnet).
7. Remove the sample tube(s) from the magnet. Add 1.0 mL of E Buffer and
vortex for 10-15 seconds
to re-suspend the IMS beads. 8. Repeat steps 5 to 7, 2 more times (a total of
3 washes). 9. After the final wash step, reconstitute the sample with 1.0 mL
of E Buffer and vortex 10-15 seconds to
mix. 10. Plate 0.1 mL of the bead suspension into mRBA plates. In addition,
plate 1:10 and 1:100 dilutions of
the IMS beads (sample) diluted in E Buffer. Use a hockey stick, swab or
spreader to spread plate the beads. Incubate for 20-24 hours at 35 +/- 2 °C.
11. Acid Treatment: for each sample, transfer 450 µL of the undiluted bead
suspension (step 9) to a microcentrifuge tube. Add 25 µL of 1N HCl and vortex
for 10-15 seconds (pH should be 2.0-2.5). 12. Place tube(s) in tube rotator
and rotate for 1 hour at 18-30 °C. 13. Dilute the IMS bead suspension by
adding 475 µL of E Buffer. 14. Vortex for 10-15 seconds to re-suspend beads
and plate 0.1 mL of the bead suspension into mRBA plates. In addition, plate a
1:10 dilution of the IMS beads (sample) diluted in E Buffer. Use a hockey
stick, swab or spreader to spread plate the beads. Incubate for 20-24 hours at
35 +/- 2 °C.
NOTE: All mixing sticks, tubes, etc. should be sterile. Separation Columns
and Separation Magnets (MACS, Miltenyi Biotec) can also be used, pleas e refer
to procedure described in the USDA-FSIS Microbiology Laboratory Guidebook
(MLG) Chapter 5.
E. Identification Colonies are picked from RBA plates and tested for
agglutination with antisera specific for the serogroup of interest (at this
stage the target serogroup is known). At least one colony of each
morphological type on each plate is tested using O145 latex agglutination kits
(Gold Standard Diagnostics PN 541050). A minimum of five latex positive
colonies from each plate are streaked onto SBA plates and incubated at 35 +/-
2 °C for 1824 hours. Colonies are then confirmed by specific latex
agglutination test.
F. Interpretation of Results/Additional Analysis Positive samples must be
confirmed as described in the USDA-FSIS test protocol described in the USDA-
FSIS Microbiology Laboratory Guidebook (MLG) Chapter 5 “Detection, Isolation
and Identification of Top Seven Shiga-Toxin Producing Escherichia coli (STECs)
from Meat Products and Carcass and Environmental Sponges”.
G. References (1) Fratamico, P., Bagi, L., Cray, W. Jr., Narang, N., Yan, X.,
Medina, M., Liu, Y. Detection by Multiplex Real-Time Polymerase Chain Reaction
and Isolation of Shiga Toxin-Producing Escherichia coli Serogroups O26, O45,
O103, O111, O121 and O145 in Ground Beef. Foodborne Pathogens and Disease
8(5):601-607. (2) USDA-FSIS Microbiology Laboratory Guidebook (MLG) Chapter 5
“Detection, Isolation and Identification of Top Seven Shiga-Toxin Producing
Escherichia coli (STECs) from Meat Products and Carcass and Environmental
Sponges”.