ABSTRACT 3M Company, St. Paul, MN, United States: Julie Yang

P2-05: Performance of the 3M Molecular Detection Assay
™
Salmonella and the 3M Molecular Detection Assay Listeria
in Environmental and Primary Production Samples
™
3M Company, St. Paul, MN, United States: Julie Yang, Cynthia Zook, Micki Rosauer
ABSTRACT
Introduction: Testing for Salmonella and Listeria is a critical component
of food safety programs as infection by these pathogens can result in
significant adverse health conditions and economical losses. The
3M™ Molecular Detection Assay Salmonella and the 3M™ Molecular
Detection Assay Listeria were developed for the rapid and specific
detection of these contaminants in samples after enrichment.
Purpose: To evaluate the performance of the new 3M methods in
a variety of environmental and primary production samples.
conducted: Set 1) where one of the duplicates was enriched blank and
one was artificially contaminated with ~10 CFU of the target organism;
and Set 2) where single samples were enriched blank to evaluate for
native contamination. Enrichments were tested using the 3M Detection
Molecular Assays and selective and differential agar and/or quantitative
PCR. A Chi square (X2) test was used to compare the results for
significant differences.
Results: Compared to agar or q-PCR, accuracy, specificity and
sensitivity were: 99%, 100% and 95% respectively for the 3M
Molecular Detection Assay Salmonella and 95.3%, 100%, 94.9%
Methods: More than 550 environmental and primary production
samples were collected in duplicates. Two sets of testing were
respectively for the 3M Molecular Detection Assay Listeria. No significant
differences were observed between the 3M method results and the
chromogenic agar results or q-PCR results as indicated by a X2 value of
less than 3.84 for either analyte.
Significance: The new methods were determined to be reliable and
accurate. For all samples evaluated, the 3M Molecular Detection Assay
Salmonella and the 3M Molecular Detection Assay Listeria demonstrated
comparable results to the other methods for the rapid, automated
detection of these organisms.
METHODS
The 3M Molecular Detection Assays are used with the 3M™ Molecular
Detection System for the rapid and specific detection of pathogen
in enriched food, feed and food process environmental samples.
The 3M Molecular Detection Assays use isothermal amplification of
nucleic acid sequences with high specificity, efficiency and rapidity,
and bioluminescence to detect the amplification. Presumptive
positive results are reported in real-time while negative results are
displayed after the assay is completed.
4. F or samples collected with Neutralizing Buffer, a portion of enriched sample was diluted 1:2 into the appropriate type of sterile
3M enrichment broth before testing using the 3M Molecular Detection System.
5. Enrichments were also either streaked onto chromogenic agar plates or tested by q-PCR, as reference points. A Chi-square (X2) test was
used to compare the results for significant differences.
6. Calculation and Interpretation: Accuracy, specificity and sensitivity of the 3M Method.
1.Cultures: Target cultures (Table 1) were propagated in 3M™ Buffered
Peptone Water ISO (BPW ISO) enrichment broth for ~18 hours at
37°C (Salmonella) or in 3M™ Modified Listeria Recovery Broth
(mLRB) for ~24 hours at 37°C (Listeria monocytogenes). To verify
spike levels used to inoculate the samples, cultures were enumerated
using 3M™ Petrifilm™ Aerobic Count Plates.
Table 1. Study Test Strains List
Genus, Species/Serotype
Salmonella Typhimurium
Responses
Chromogenic agar and qPCR Positive (R+)
Chromogenic agar and qPCR Negative (R–)
3M Method Positive (A+)
+/+ Positive Agreement (PA)
R–
–/+ Positive Deviation (PD) as —
A+
3M Method Negative (A–)
A–
+/– Negative Deviation (ND) as —
R+
( )
( )
–/– Negative Agreement (NA)
Accuracy (AC), Specificity (SP) and Sensitivity (SE) of the 3M method were calculated as follows (where N=total number of samples):
(PA+NA)
NA
PA
AC =
x 100% SP =
x 100% SE =
x 100%
N
(PD+NA)
(PA+ND)
ATCC
14028
Salmonella Enteriditis
13076
Salmonella — Environmental and Poultry Rinse Studies
Salmonella Newport
Listeria monocytogenes
Listeria monocytogenes
Listeria monocytogenes
6962
7644
19118
43256
142 different environmental samples, including poultry drag swabs, were collected in duplicate from various farms. Surfaces tested included concrete
floors and ceiling, wooden roosts, metal feeders, and stainless steel equipment and pipes. 84 bird rinses were provided by 9 different poultry processing
plants. Table 2 indicates matrices, sampling devices and enrichment volume of 3M BPW ISO used.
ATCC — American Type Culture Collection
2. Sample enrichments were performed for Salmonella in 3M BPW
ISO at 37°C and Listeria in 3M mLRB at 37°C. All enrichment broths
were pre-warmed to the incubation temperature prior use.
3. 3M Molecular Detection Assay: Each enrichment was extracted
and an aliquot of the lysate was tested using the 3M Molecular
Detection System (Figure 1).
3M Method Listeria:
Sample enrichment
Incubation (24 –28 hours)
3M BPW ISO Enrichment Volume
30mL
3M™ Dry-Sponge
Surfaces
10mL D/E Neutralizing broth
50mL
3M™ Dry-Sponge with String
Poultry fecal drag swab
10mL BPW
50mL
Listeria — Environmental Samples Studies
LS
431 environmental samples were collected in duplicates from dairy farms, cattle farms and a ready-to-eat (RTE) food processing plant, where sampling
locations were chosen specifically to increase the likelihood of finding naturally-occurring Listeria. Table 3 summarizes surfaces tested, sampling devices
and enrichment volume of 3M mLRB used.
Transfer 20µL enriched
sample to lysis tube.
Table 3. Surfaces and Collection Devices Summary
Sampling Devices
Surface Material
3M™ Dry-Sponge
Stainless steel
0-20ºC
99-101ºC
Cool 10 minutes on chill block.
Let sit 5 minutes.
Heat 15 minutes at 100°C (±1°)
20µL
Transfer 20µL lystate to reagent
tubes containing lyophilized pellet.
Place Speed
Loader Tray into
instrument. Close
the lid and start run.
Hydrating Solution
BPW
Two sample sets were tested as follows (All enrichments were performed at 37°C for 18 hours):
Set 1: Suspensions of Salmonella Typhimurium, S. Enteritidis or S. Newport were used to artificially contaminate one of the duplicates with ~10 CFU.
The other duplicate was enriched blank.
Set 2: Single samples enriched as blank to evaluate for native contaminations.
Figure 1. Simplified Protocol for Productivity
3M Method Salmonella:
Sample enrichment
Incubation (18 –24 hours)
Table 2. Matrices and Collection Devices Summary
Sampling Devices
Sample Type
None
Poultry carcass rinse
Transfer closed tubes
to Speed Loader Tray.
Performs amplicifations in
75 minutes. Automated and
color coded real-time results.
TTR:
Neutralizing or Hydrating Solution
D/E Neutralizing broth
3M mLRB Enrichment Volume
225mL
3M™ Dry-Sponge
Concrete
D/E Neutralizing broth
225mL
3M™ Dry-Sponge
3M™ Sponge-Stick
3M™ Quick Swab
3M™ Enviro Swab
3M™ Enviro Swab
Plastic
Various surfaces
Metal drains
Plastic piping
Various surfaces
Neutralizing buffer
D/E Neutralizing broth
Letheen broth
Modified Letheen broth
Modified Letheen broth
225mL
225mL
10mL
10mL
10mL
Three sample sets were tested as follows:
Set 1: Paired samples were incubated blank, or were spiked with suspensions of L. monocytogenes at a level of ~10 CFU per sponge, then incubated for
22 hours at 37°C.
Set 2: Paired samples were incubated blank, or were spiked with suspensions of L. monocytogenes at a level of ~10 CFU per sponge, then incubated for
24 hours at 37°C.
Set 3: Single samples collected from a RTE food processing plant were enriched blank for 22, 26 and 28 hours at 37°C to evaluate for native contamination.
20 –30 hours
RESULTS
CONCLUSIONS
No significant differences were observed between the 3M method results and the chromogenic agar results or q-PCR results as indicated by a X2 value
of less than 3.84 for either analyte (Table 4).
A Chi square (X2) of ≥ 3.84 indicates that the proportions positive for the different methods differ significantly at p ≤ 0.05. This criterion is used by AOAC
International for analysis of confirmed positives; here it used for analysis of presumptive positive results.
The new 3M Molecular Salmonella and Listeria spp. methods were
determined to be reliable and accurate, and demonstrated comparable
results to chromogenic agar and/or q-PCR results. The 3M methods
were shown to be compatible with a variety of environmental samples,
including poultry drag swabs, bird rinses and fecal containing samples.
In addition, the process flow was more streamlined than the q-PCR
method and faster than the cultural methods.
Table 4. Performance Results Summary
Target
Type of Sample
Salmonella
Environmental
Salmonella
Bird Rinse
Listeria
Environmental
n
142
84
431*
Accuracy
99%
97.6%
95.3%
Specificity
100%
100%
100%
Sensitivity
95%
93.9%
94.5%
*All samples combined (n = 431, incubation time 22, 24, 26 or 28 hours)
References
a. Feldsine, Abeyta and Andrews, 2002. Section 5.3.1 Test for Significant Difference (X2), Journal of AOAC, Vol. 85, No. 5, P. 1187-1200.
X2
0.00
1.33
1.8
ACKNOWLEDGEMENTS
The authors thank Molly Rother, Amber Turner and Olivia Trittipo
for their dedicated laboratory contributions.
© 3M 2012. 3M and Petrifilm are trademarks of 3M.