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of Shiga-toxins using the USDA approved Real-Time PCR assay. Final biochemical
confirmations were obtained by VITEK 2 GN Biochemical Identification following AOAC
OMA 2011.17.
USDA/FSIS-MLG 5B.05: Detection and Isolation of non-O157 Shiga Toxin-Producing
Escherichia coli (STEC) from Meat Products and Carcass and Environmental Sponges
All raw beef trim test portions were prepared as outlined in the study protocol. Following
equilibration of the inoculum in the matrix, 325 ± 32.5 g test portions were enriched in 975
± 19.5 mL of pre-warmed mTSB + CAA, homogenized by stomaching for 2 minutes and
incubated for 15-24 hours at 42 ± 1
o
C.
Following incubation, 30 µL of the primary enrichment from each sample was screened
using a USDA/FSIS-MLG 5B.05 approved commercially available Real-Time PCR assay
(DuPont Nutrition and Health, BAX System). Each sample was screened for the presence
of STEC virulence factors Shiga-like toxin 1 and/or Shiga-like toxin 2 (
stx
1
/stx
2) and intimin
(
eae
). If a sample produced positive results for
stx
1 or
stx
2 and
eae,
an additional screen
for the targeted sixserogroups (O26, O45, O103, O111, O121 and O145) was conducted.
Regardless of the screening result, all samples were subjected to isolation by
immunomagnetic separation (IMS) in a ferromagnetic column with paramagnetic beads by
transferring a 1.0 mL aliquot of the primary enrichment to microcentrifuge tubes containing
a 50 µL suspension of
E. coli
O26 immunomagnetic beads (Abraxis, Inc.). The
microcentrifuge tubes were placed on a Labquake agitator and rotated for 15 minutes. After
rotation, the bead and sample solution was transferred to a MACS large cell separation
column and was washed four times with 1 mL of E buffer before collecting 1 mL of the final
elute in a sterile tube.
Following the IMS procedure, a 1:10 and 1:100 dilution of each IMS suspension in E Buffer
was spread-plated on mRBA. A 450 µL aliquot of each remaining sample was transferred
to a microcentrifuge tube and mixed with 25 µL of 1 N HCl. The microcentrifuge tubes
were gently mixed and placed on a Labquake agitator and rotated for 1 hour. After rotating,
475 µL of E buffer was added to each sample tube. The acid-washed IMS suspension and
a 1:10 dilution of the acid-washed IMS suspension in E Buffer were plated on mRBA. All
mRBA plates were incubated for 20-24 hours at 35 ± 1°C. After incubation, any mRBA
plates containing typical colonies were tested by serogroup specific latex agglutination. Up
to 5 isolated colonies were streaked to SBA and incubated for 16-24 hours at 35 ± 1°C.
After incubation, SBA plates were observed for purity. Isolated colonies from SBA were
confirmed positive for the presence of
stx
1 or
stx
2 and
eae
by Real-Time PCR. The
serogroup was confirmed by latex agglutination and by Real-Time PCR. Final biochemical
confirmations were obtained by VITEK 2 GN Biochemical Identification following AOAC
OMA 2011.17.
mericon
E.coliO157Screen Plus and
mericon
E.coliSTEC O-Type
Pathogen Detection
Assay
For raw ground beef and raw beef trim, all test portionswere assessed concurrently with
the reference methods. The paired325 g test portions were enriched in 975 mLof pre-
warmed (42 ± 1°C) mTSB+CAA,homogenized by stomaching for 2 min± 10 sec, and
incubated for 10± 1hr at 42 ± 1°C.
OMAMAN-36 E/ AOAC PTM Report
ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use O ly