AOAC-RI ERP Book MICRO.pdf

CONFIDENTIAL INFORMATION

Page 11 of 13

4.0 DISCUSSION

The purpose of this study was to validate the

3M™

Molecular Detection Assay

(MDA) for detection of

Escherichia coli

O157:H7 in ground beef with an 18 hr

enrichment time. This study followed the format for the validation of a candidate

method using the experimental setup and Probability of Detection (POD) statistical

analysis provided in the February 24, 2012 version of the AOAC International

Methods Committee Guidelines for Validation of Microbiological Methods for Food

and Environmental Surfaces document (1). Portions of ground beef (325 g sample

size) were inoculated with

E. coli

O157:H7 at a concentration aimed to achieve a

targeted level of contamination. The inoculation levels targeted were chosen in

order to meet the targeted POD values for the different inoculation levels. The

concentrations achieved through inoculation were found to be 1.739 x 10

-3

MPN/g

for the low inoculation level of ground beef and 1.538 x 10

MPN/g for the high

inoculation level (Table 1). In addition to this, the AOAC fractional positive criterion

(POD between 0.25 and 0.75) was met, providing evidence that the inoculation

procedure was successful for ground beef with an 18 hr enrichment time (Table

4).

Review of the POD calculation outcomes for the 18 hr ground beef enrichment

candidate method (325 g Ground Beef Analyzed via the 3M

MDA) and reference

method (325 g Ground Beef Analyzed via the USDA FSIS MLG Method 5.09)

shows that the 95% confidence intervals contained 0.000 at all inoculation levels

for the dPOD

, comparing the ‘screening stage’ and ‘confirmation stage’ of the

candidate method, and the dPOD

, comparing the candidate method and

reference method (Table 5). Because of this, for the candidate method, the POD

values were not significantly different from their corresponding POD

values.

Based on this outcome, it can be stated that the probability of detecting a positive

result in the “screening stage” of the candidate method was not significantly

different from the probability of detecting a positive result in its “confirmation stage”

at any of the inoculation levels tested. Additionally, for the candidate method

versus the reference method, the POD

values were not significantly different from

their corresponding POD

values. Based on these outcomes, by fulfilling the

criteria to contain 0.000 within the 95% confidence intervals, it can be stated that

the probability of detecting a positive result in the candidate method was not

significantly different from the probability of detecting a positive results in the

reference method. Thus, the candidate method and the reference method can be

considered equivalent, based on the AOAC International guidelines. Also, the lack

of false positive or false negatives (Table 4

) when comparing the ‘screening stage’

and the ‘confirmation stage’ of the candidate method further supports the

adequacy of the method.