AOACRIMicroMethods-2017Awards

allace

et al

ournal of

AOAC I

nternational

97, N

. 3, 2014

869

ground beef with soy (85% lean), beef trim, frankfurters (beef),

shrimp, ground turkey, chicken wings, dried eggs (whole,

powdered), shell eggs, frozen peas, orange juice, instant nonfat

dry milk, ice cream (12% fat), peanut butter (52% fat), cocoa

(unsweetened), white pepper, milk-based infant formula,

ceramic tile, and plastic surfaces. The results obtained using the

test method indicate no statistical difference with the reference

method when compared to the corresponding reference method

results (Appendix

3).

In addition, two of the precollaborative study matrixes—

frankfurters (pork plus turkey) and orange juice (pasteurized

not-from-concentrate)—were evaluated in a total of

independent laboratories as part of the collaborative study

to demonstrate repeatability and reproducibility of the internal

laboratory results independent of the end user. The results

obtained using the BAX System method indicate no statistical

difference when compared to the corresponding reference

method results.

Collaborative Study

Study Design

Collaborators analyzed two representative matrixes (pork

and turkey frankfurters and pasteurized, not-from-concentrate

orange juice without pulp), 12

replicate test portions from each

of three contamination levels (low, high, and uninoculated),

comparing the performance of the BAX System Real-Time

PCR Assay for

Salmonella

to appropriate reference culture

methods. A total of 15

laboratories participated in the study, with

laboratories reporting data for each matrix. Each collaborator

received instructions for performing the study and required

materials prior to the start of the study. If necessary, training

on the BAX System was provided to laboratory personnel by a

DuPont representative.

The collaborative study was conducted in accordance with

the

AOAC INTERNATIONAL Methods Committee Guidelines

for Validation of Microbiological Methods for Food and

Environmental Surfaces

, Appendix J

(4). Frankfurter samples

were evaluated against the USDA-FSIS MLG reference method

as a paired study, as the test and reference method enrichment

protocols are identical. Orange juice samples were evaluated

against the FDA-BAM reference method as an unpaired study, as

the BAX System method uses enrichment in proprietary media.

Estimates of repeatability, reproducibility, and probability of

detection (POD) were evaluated.

Preparation of Inocula and Test Portions

Sample product was obtained from a local retail outlet

and screened by the organizing laboratory to identify any

naturally contaminating

Salmonella

and determine a total

aerobic plate count. For each sample type, five analytical size

portions (25

g for orange juice and 325

g for frankfurters)

were screened for

Salmonella

using the appropriate reference

method. Although naturally contaminated samples would have

been preferred, all samples tested returned negative results for

Salmonella

. Therefore, each sample matrix was artificially

inoculated with a different serovar of

Salmonella

for use in this

study.

Portions of each sample type were inoculated at levels that

on the day of initiation of analysis produced a high spike level

(POD approximately 1.0 or approximately 5 CFU/test portion)

and a low spike level (POD 0.25–0.75 or 0.2–2.0

CFU/test

portion). Additional matrix was left uninoculated to serve as

negative controls.

To inoculate frankfurter samples, a pure colony of

Salmonella

Typhimuriumwas transferred fromTrypticase Soy agar with 5%

sheep’s blood (SBA) into Brain Heart Infusion (BHI) broth and

incubated at 35°C for 18–24

h. The inoculum was heat stressed

in a 55°C water bath for 10 min to obtain a percent injury of

approximately 70% as determined by plating onto selective

Xylose Lysine Desoxycholate (XLD) agar and nonselective

TSA. Four portions of equal size were inoculated drop-wise

with an 18–22

h culture of the target organism, and then

homogenized by hand. All four portions were combined one at

a time into a single container, homogenizing the bulk material

after each portion was added. The bulk lot was separated into

two sampling containers and 40

samples (20 for each method)

weighing 25

g each were removed from each container. Each

g sample was combined with 300

g uncontaminated matrix

to create 325

g test portions. The remaining spiked matrix was

rehomogenized by combining the material from both containers

into one and mixing thoroughly for the purposes of maintaining

an even distribution of the organism.

To inoculate orange juice, a pure colony of

Salmonella

Hadar

was transferred from SBA into BHI broth containing 1% glucose

and incubated at 35°C for 18–24

h. This stress protocol resulted

in a percent injury of approximately 60% (as determined by

plating onto selective XLD agar and nonselective TSA). The

inoculum was added drop-wise to a bulk quantity of orange

juice to reach the desired contamination level, and then mixed

to achieve equal distribution of the inoculum throughout. This

spiked bulk quantity was divided into 25 mL test portions for

analysis.

Test Portion Distribution

All test portions were randomized and blind-coded by

the organizing laboratory, then shipped overnight to each

collaborating laboratory and maintained at 2–8°C until they

were analyzed. The total hold time of samples was 48

h for

frankfurters and 96

h for orange juice, including shipment

time to each participating laboratory. On the first day of test

sample analysis, a 5-tube, 3-level most probable number

(MPN) estimation of contamination levels was conducted

by the organizing laboratory using the appropriate reference

method. The Least Cost Formulations, Ltd (Norfolk, VA) MPN

Calculator-Version 1.6

(5) was used to determine the MPN

values and 95% confidence intervals. The MPN is reported for

each level of each matrix in Appendix 4, Tables

1–6 as MPN/

test portion with 95% confidence intervals.

Test Portion Analysis

For testing frankfurters, each collaborator received

12 low-spike, 12 high-spike, and 12 uncontaminated 325

test portions, blind-coded so that the contamination level

was unknown to the collaborator. Approximately one-third

to one-half of 2925 ± 58.5

mL of sterile buffered peptone

water (BPW) was added to each portion, and each portion

was homogenized approximately 2 min. The remainder of the