216 / 453
4
the temperature of this portion upon receipt of the package, document the results on the Sample
1
Receipt Confirmation form provided and fax or email it back to the study director.The shipment
2
and hold timesof the inoculated test material had been verified as a quality control measure prior
3
to study initiation.
4
5
Test Portion Analysis
6
7
Collaborators were instructed to follow the appropriate preparation and analysis as outlined in
8
the study protocol. A single set of test portions (36) was enriched following a paired study
9
design with the candidate method utilizing two sample preparation techniques, manual and
10
automated DNA extraction methods. Each collaborator received 36 test portions (12 high, 12 low
11
and 12 un-inoculated controls). Thetest portions (325 g) were enriched with 975 mL of modified
12
tryptic soy broth(includes casamino acids), homogenized for 2 minutes and incubated for 10
13
hours at 42 ±1
o
C. After 10 hours, a subsample of each enrichment was removed for analysis by
14
the
mericon
methods and the remaining sample was incubated for an additional 5-14 hours (a
15
total incubation time of 15-24 hours) at 42 ± 1
o
C.
16
17
Following the 10 hour enrichment, samples were assayed by the
mericon
E. coli O157Screen Plus
18
Pathogen Detection Assay(using both the manual and automated DNA extraction procedure
19
methods) and then by the
mericon
E. coliSTEC O-Type Pathogen Detection Assay (using both
20
manual and automated DNA extraction method procedures). Regardless of presumptive results
21
(from both the screening assay and the O-Type assay) obtained from both assays and extraction
22
procedures,all test portions were confirmed following the USDA/FSIS MLG 5.09 reference
23
method after a total incubation time of 15-24 hours, beginning with a screen using an
24
USDA/FSIS approved lateral flow device. All positive test portions were evaluated for the
25
presence of O157 and H7 antigens by latex agglutination, for the presence of
stx
1/2 and
eae
26
genes by a USDA/FSIS approved PCR test and biochemically confirmed by the API 20 E or
27
VITEK 2 GNbiochemical identification test, AOAC Official Method 2011.17[7].
28
29
Statistical Analysis
30
31
Each collaborating laboratory recorded results for both
mericon
E. coli O157Screen Plus
32
Pathogen Detection Assay and
mericon
E. coliSTEC O-Type Pathogen Detection Assay, using
33
both the manual and automated DNA extraction procedures on the data sheets provided. The data
34
sheets were submitted to the study director at the end of testing for statistical analysis. Data for
35
each contamination level was analyzed using the probability of detection (POD)statistical model
36
[8]. The probability of detection (POD) was calculated as the number of positive outcomes
37
divided by the total number of trials. The POD was calculated for the candidate presumptive
38
results, POD
CP,
the candidate confirmatory results (including false negative results), POD
CC
, the
39
difference in the candidate presumptive and confirmatory results, dLPOD
CP,
presumptive
40
candidate results that confirmed positive (excluding false negative results), POD
C,
the reference
41
method, POD
R
, and the difference in the confirmed candidate and reference methods, dLPOD
C
.
42
A dLPOD
C
confidence interval not containing the point zero would indicate a statistically
43
significant difference between the
mericon
E. coliO157 Screen Plus Pathogen Detection Assay
44
or
mericon
E. coliSTEC O-Type Pathogen Detection Assay, and the reference methodat the 5 %
45
probability level. In addition to POD, the repeatability standard deviation (s
r
), the among
46
laboratory repeatability standard deviation (s
L
), the reproducibility standard deviation (s
R
)and the
47
P
T
value were calculated. The s
r
provides the variance of data within one laboratory, the s
L
48
provides the difference in standard deviation between laboratories and the s
R
provides the
49
OMAMAN-36 A : Collaborative Study Manuscript
For ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only