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2
factor intimin
eae
[1]. Depending on the reference cited, there are approximately 200 to 400
1
STEC serotypes, many of which have not been implicated in human illness; however, a subset of
2
STEC called enterohemorrhagic
Escherichia coli
(EHEC) includes only those that cause serious
3
illness [2]. EHEC outbreaks have been traced to many different types of foods such as the
4
following: raw ground meats, unpasteurized (“raw”) milk, unpasteurized fruit juice, lettuce,
5
spinach, sprouts, and more recently, commercially manufactured frozen cookie dough. Infections
6
caused by EHEC can progress into life-threatening problems with children and immuno-
7
compromised people being at especially high risk[1].
8
9
QIAGEN’s
mericon
E. coli O157 Screen Plus and
mericon
E. coliSTEC O-TypePathogen
10
Detection Assaysaremultiplex PCR assays designed to provide both a screening and
11
confirmation assay. The
mericon
E. coliO157 Screen Plus is designed to detect the presence of
E.
12
coli
O157 and pertinent virulence genes
stx
1/2 and
eae.
The
mericon
E. coliSTEC O-Typeis
13
designed to confirm the presence of
E. coli
O157:H7 and the big six non-O157 STEC strains (
E.
14
coli
O26, O45, O103, O111, O121 and O145). The assays can be utilized with DNA extracted
15
following a manual or automated procedure on the QIAsymphony QS/AS instrument. Final
16
prepared PCR samples are analyzed using the Rotor-Gene Q cycler and its software.
17
Prior to the collaborative study, both the
mericon
E. coliO157 Screen Plus and
mericon
E.
18
coliSTEC O-Type Pathogen Detection Assays with manual and automated DNA extractionwere
19
validated according to current AOAC Guidelines[3] in a harmonized AOAC
®
Performance
20
Tested Method
SM
(PTM) study. The objective of thestudy was to demonstrate that the
mericon
21
methods could detect and confirm the presence of both
E. coli
O157 and non-O157 STECin
22
select food matrices as claimed by the manufacturer. The assays, using both the manual and
23
automated DNA extraction procedures, were evaluated for 3 matrices:fresh spinach (25g), raw
24
ground beef (73% lean) (325g)and raw beef trim (325g).
25
26
Additional PTM parameters (inclusivity, exclusivity, ruggedness, stability and lot-to-lot
27
variability) were tested and satisfied the performance requirements for AOAC PTM approval.
28
The
mericon
E. coli O157Screen Plus Pathogen Detection Assay was awarded PTM certification
29
number 101503 and the
mericon
E. coli STEC O-Type Pathogen Detection Assay was awarded
30
PTM certification number 101504 on October23
rd
, 2015.
31
32
The purpose of this collaborative studywas to compare the reproducibility of both the
mericon
E.
33
coliO157 Screen Plus Pathogen Detection Assay and
mericon
E. coliSTEC O-Type Pathogen
34
Detection Assay with manual and automated DNA extraction method to the United States
35
Department of Agriculture (USDA) Food Safety Inspection Service (FSIS) -Microbiology
36
Laboratory Guidebook (MLG)Chapter 5.09
Detection, Isolation and Identification of
37
Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges
[4] for
38
73% lean raw ground beef (325 g).
39
40
Collaborative Study
41
42
Study Design
43
44
In this collaborative study, one matrix, raw ground beef (73% lean), wasevaluated. The matrix
45
was obtained from a local retailer and screened for the presenceof
Escherichia coli
O157by the
46
USDA/FSIS MLG 5.09 reference method
.
The raw ground beef was artificially contaminated
47
with fresh unstressed cells of
Escherichia coli
O157:H7, American Type Culture Collection
48
(ATCC) 43895,at two inoculation levels: a high inoculation level of approximately 2-5 colony-
49
OMAMAN-36 A : Collaborative Study Manuscript
For ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only