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± 2°C for 24 ± 2 hours. Growth from the TSTB was used to conduct the flagellar H serological test. Growth from
samples producing typical biochemical reactions in TSI and LIA, were streaked to TSA slants and incubated at 35 ±
2
o
C for 18-24 hours. Growth from the TSA slant was used to conduct the polyvalent O serological test for
biochemical confirmation and final confirmations using VITEK
®
2 GN following AOAC Official Method 2011.17.
Inclusivity and Exclusivity
The inclusivity/exclusivity evaluation consisted of analyzing 105 different
Salmonella
serotypes (Table P) and 31
closely related non-target isolates (Table Q) by the 3M MDA
Salmonella
. There were two separate inclusivity and
exclusivity procedures conducted using the two new modified protocols. For both procedures, all exclusivity
organisms were cultured in brain heart infusion (BHI) for 24 hours at conditions that would promote optimal
growth. For the first modified protocol, all
Salmonella
isolates were cultured in 3M-BPW and incubated at
41.5±1°C for 18 hours. Following incubation,
Salmonella
cultures were diluted in phosphate buffered water (PBW)
to 100 x the limit of detection (LOD) while exclusivity cultures were analyzed without dilution. For the second
modified protocol, the
Salmonella
cultures were transferred from an 18-hour enrichment in BPW into RV 10,
incubated in a water bath for 4 hours, and then diluted in BPW to 100 times LOD. For both procedures, all
exclusivity organisms were cultured in brain heart infusion (BHI) for 24 hours at conditions that would promote
optimal growth. After incubation inclusivity and exclusivity test cultures were randomized, blind coded and
analyzed according to the 3M MDA
Salmonella
.
Results
Method Comparison
For the high inoculum level, low inoculum level, and the un-inoculated control level, the probability of detection
(POD) was calculated as the number of positive outcomes divided by the total number of trials. The POD was
calculated for the candidate presumptive results, POD
CP,
the candidate confirmatory results, POD
CC
, the difference in
the candidate presumptive and confirmatory results, dPOD
CP,
the reference method, POD
R
, and the difference in the
confirmed candidate and reference methods, dPOD
C
. POD analyses were conducted for all time points of the 3M
MDA
Salmonella
and for the reference methods. Results of the method comparison and detailed POD results are
presented in the Appendix: Tables D through M for the internal study and Tables N & O for the independent study.
As per criteria outlined in Appendix J of the AOAC Microbiology guidelines
4
, fractional positive results were
obtained for all matrices analyzed.
Internal Study
Raw Ground Beef – 25 g
For the low inoculation level at 10, 12 and 18 hours, there were 14 presumptive and confirmed positives for the
candidate method and 14 confirmed positives for the USDA/FSIS reference method. A POD
C
value of 0.70 was
obtained with 95% confidence intervals of (0.48, 0.85). A POD
R
value of 0.70 was obtained with 95% confidence
intervals of (0.48, 0.85). A dPOD
C
value of 0.00 was obtained with 95% confidence intervals of (-0.27, 0.27),
indicating no statistical difference between the candidate and reference method. For the low inoculation level at each
time point a POD
CP
value and a POD
CC
value of 0.70 was obtained with 95% confidence intervals of (0.48, 0.85). A
dPOD
CP
value of 0.00 with 95% confidence intervals of (-0.27, 0.27) indicating no statistical difference between the
candidate method presumptive and confirmed results.
For the high inoculation level at 10, 12 and 18 hours, there were 5 presumptive and confirmed positives for the
candidate method and 5 confirmed positives for the reference method. A POD
C
and POD
R
value of 1.00 was
obtained with 95% confidence intervals of (0.57, 1.00). A dPOD
C
value of 0.00 was obtained with 95% confidence
intervals of (-0.43, 0.43), indicating no statistical difference between the candidate and reference method. A POD
CP
value and a POD
CC
value of 1.00 was obtained with 95% confidence intervals of (0.57, 1.00). A dPOD
C
value of
0.00 was obtained with 95% confidence intervals of (-0.43, 0.43), indicating no statistical difference between the
candidate method presumptive and confirmed results.
Raw Ground Beef – 325 g
2013.09 /PTM Matrix Extension Report
March 2014
Expert Review Panel Use Only
AOAC Research Institute
Expert Review Panel Use Only