This document describes a study that evaluated a new multiplex quantitative PCR (qPCR) assay for detecting Yersinia pestis in meat products. The study artificially contaminated hot dogs, baby food, and ground beef with low and high levels of Y. pestis. Samples were tested using both the new qPCR assay and culture methods. The qPCR assay detected Y. pestis in all samples at high levels, while detecting it in more samples at low levels compared to culture. The qPCR assay showed potential for rapid detection of Y. pestis in difficult food matrices.

1. RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
Plague, caused by Yersinia pestis, has had devastating effects on the human population
throughout our history. Disappearance of this disease is unlikely due to the wide range of
mammalian hosts and their attendant fleas, which are vectors of the causative agent. Y. pestis
was first isolated by Alexandre Yersin in 1894 in Hong Kong. Since its discovery, Yersinia pestis
has been isolated from all continents. Rapid detection of high priority agents such as Yersinia
pestis from difficult food matrices is essential in preventing a foodborne outbreak of the
disease. Meat products, especially, can be a challenging matrix for sensitive detection and
isolation of pathogens at low levels.
INTRODUCTION
The objective of the present study was to evaluate and validate a new multiplex qPCR
screening assay for the detection of Y. pestis in meat products.
PURPOSE
DNA isolation: was prepared from 24 hours enrichment using MagNA Pure Compact Nucleic Acid
Isolation Kit I - Large Volume (Roche REF 03 730 972 001) in accordance with the manufacturer
instructions. 2µl of the template was used in an FDA Y. pestis multiplex qPCR protocol.
MATERIALS AND METHODS RESULTS
All samples were positive by both methods, no difference (p < 0.05) between detection rates
using either qPCR method or culture was observed at the high spike levels. In the screening of
frozen beef patties, at low levels (110 cfu/g), all spiked samples tested positive (6/6) by qPCR
compared to (4/6) by the culture method. However, fractional qPCR detection (all gene
targets) and cultural recovery rates were observed at low level inoculums for hot dog and baby
food (4/6 and 2/6, respectively).
DISCUSSION
REFERENCES
Roumen Penev1, Karen Jinneman2, Benjamin Alonzo1 and Ken Yoshitomi2
(1)Arizona State Public Health Laboratory Phoenix, AZ
(2)U.S. Food and Drug Administration, Pacific Regional Laboratory NW, Applied Technology Center, Bothell, WA
Rapid qPCR Detection and Cultural Isolation of Yersinia pestis in Artificially Contaminated Meat Products
MATERIALS AND METHODS
Sample inoculation: Hot dog and meat-based baby food were artificially contaminated with Y.
pestis at the following levels: un-inoculated, low (~1 cfu/g) and high (~10 cfu/g) with six
replicates per level (n = 6/level). Ground beef patties were inoculated at 10-fold greater
concentrations, based on earlier experimental data. Four additional food matrices (deli meats,
ground beef, ground turkey and raw chicken from 20 different sources each) were surveyed by
qPCR and culture at both inoculated (3-390 cfu/g) and un-inoculated levels
GROUND TURKEY DELI MEAT RAW CHICKEN Ground Beef
3 cfu/g 390 cfu/g 300 cfu/g 300 cfu/g
Cycle Threshold (Ct) Cycle Threshold (Ct) Cycle Threshold (Ct) Cycle Threshold (Ct)
SC II AB 7500 SC II AB 7500 SC II AB 7500 SC II AB 7500
ypchr lcrV pla A IC ypchr lcrV pla A IC ypchr lcrV pla A IC ypchr lcrV pla A IC ypchr lcrV pla A IC ypchr lcrV pla A IC ypchr lcrV pla A IC ypchr lcrV pla A IC
1 0.00 0.00 39.62 25.37 34.90 0.00 34.56 26.01 26.97 0.00 25.26 29.16 27.61 0.00 25.03 26.93 0.00 0.00 0.00 25.19 0.00 0.00 31.37 25.51 23.42 0.00 23.05 25.11 23.12 0.00 21.73 25.56
2 36.89 0.00 35.34 25.20 33.79 0.00 32.51 26.14 24.90 0.00 23.10 35.94 25.55 0.00 23.12 26.83 0.00 0.00 30.60 24.77 0.00 0.00 27.03 25.48 25.64 0.00 25.52 25.13 25.57 0.00 24.36 25.49
3 0.00 0.00 0.00 25.32 0.00 0.00 0.00 25.80 25.88 0.00 24.41 30.35 26.42 0.00 24.55 26.88 0.00 0.00 0.00 25.35 0.00 0.00 34.51 25.29 30.34 0.00 29.95 25.15 31.12 0.00 28.90 25.46
4 35.94 0.00 38.36 25.32 34.48 0.00 32.74 25.97 28.66 0.00 26.79 28.61 28.57 0.00 26.19 26.77 28.62 0.00 26.64 25.19 27.80 0.00 25.16 25.40 27.37 0.00 26.18 25.19 27.20 0.00 25.07 25.52
5 27.31 0.00 26.08 25.25 27.98 0.00 25.18 25.96 25.52 0.00 24.61 31.72 26.85 0.00 24.69 26.74 19.08 0.00 18.07 0.00 18.70 0.00 16.98 25.23 0.00 0.00 28.44 25.21 33.43 0.00 27.23 25.50
6 27.10 0.00 25.61 25.36 26.00 0.00 23.09 25.96 26.58 0.00 25.23 29.60 26.96 0.00 24.52 26.64 0.00 0.00 0.00 25.64 0.00 0.00 0.00 25.31 0.00 0.00 28.69 25.38 31.21 0.00 27.29 25.55
7 29.00 0.00 27.75 25.34 29.11 0.00 26.02 26.06 28.61 0.00 26.85 28.75 29.35 0.00 26.92 26.82 31.16 0.00 28.40 25.19 30.10 0.00 26.75 25.53 31.59 0.00 27.67 25.20 30.27 0.00 26.37 25.64
8 0.00 0.00 0.00 25.26 0.00 0.00 34.29 26.08 29.26 0.00 26.94 28.89 30.08 0.00 26.95 26.97 29.54 0.00 28.08 25.06 29.34 0.00 26.80 25.79 19.93 0.00 19.24 29.90 19.49 0.00 18.17 26.19
9 44.03 0.00 0.00 25.30 38.44 0.00 36.48 26.07 27.33 0.00 25.61 28.91 27.88 0.00 25.30 26.88 0.00 0.00 31.17 25.18 32.12 0.00 29.35 25.59 24.81 0.00 23.99 25.20 24.68 0.00 23.30 25.52
10 0.00 0.00 0.00 25.66 38.34 0.00 34.30 25.99 30.10 0.00 28.03 29.04 30.56 0.00 27.42 26.82 0.00 0.00 0.00 25.36 0.00 0.00 38.99 25.74 24.76 0.00 24.19 25.17 24.20 0.00 22.58 25.54
11 34.54 0.00 32.49 25.58 33.14 0.00 30.44 26.03 28.40 0.00 26.68 28.81 28.96 0.00 26.49 26.80 0.00 0.00 0.00 25.03 0.00 0.00 0.00 25.46 0.00 0.00 28.08 25.14 33.83 0.00 26.94 25.58
12 32.78 0.00 32.59 25.62 31.22 0.00 29.03 25.94 32.15 0.00 29.30 29.12 33.60 0.00 29.90 26.75 0.00 0.00 0.00 25.33 0.00 0.00 34.51 25.45 27.80 0.00 26.96 25.62 27.86 0.00 26.00 25.65
13 0.00 0.00 0.00 25.19 38.59 0.00 32.30 26.04 26.95 0.00 25.30 28.70 17.18 0.00 25.18 26.79 0.00 0.00 0.00 25.04 0.00 0.00 31.35 25.51 0.00 0.00 27.28 25.16 33.71 0.00 25.62 25.63
14 0.00 0.00 33.90 25.44 32.17 0.00 28.73 25.99 28.84 0.00 26.86 28.80 29.07 0.00 26.55 26.79 0.00 0.00 0.00 25.13 0.00 0.00 0.00 25.27 0.00 0.00 28.25 25.87 0.00 0.00 25.66 25.59
15 0.00 0.00 0.00 25.12 0.00 0.00 0.00 25.93 29.45 0.00 27.55 29.14 30.32 0.00 27.25 26.74 35.97 0.00 33.35 24.75 31.68 0.00 28.38 25.62 0.00 0.00 29.41 25.98 0.00 0.00 29.93 25.46
16 0.00 0.00 0.00 25.33 0.00 0.00 36.02 25.83 30.03 0.00 27.75 28.55 30.83 0.00 28.12 26.83 0.00 0.00 44.74 25.22 0.00 0.00 30.12 25.75 33.21 0.00 31.93 25.19 31.00 0.00 28.80 25.44
17 0.00 0.00 0.00 25.17 0.00 0.00 0.00 25.80 28.89 0.00 27.50 28.61 29.26 0.00 26.88 26.84 0.00 0.00 0.00 25.05 0.00 0.00 30.62 25.64 26.89 0.00 26.58 25.11 27.08 0.00 25.44 25.53
18 0.00 0.00 0.00 25.18 0.00 0.00 0.00 26.03 29.65 0.00 28.18 28.74 30.07 0.00 27.55 26.77 0.00 0.00 0.00 24.96 0.00 0.00 0.00 25.71 23.36 0.00 23.38 24.85 23.24 0.00 22.28 25.46
19 0.00 0.00 0.00 25.25 0.00 0.00 0.00 27.06 28.26 0.00 26.75 28.74 28.54 0.00 26.26 26.75 0.00 0.00 28.70 25.06 31.10 0.00 27.14 25.48 26.02 0.00 24.81 25.12 26.07 0.00 24.89 25.62
20 0.00 0.00 0.00 25.20 0.00 0.00 0.00 25.96 28.71 0.00 26.48 28.74 29.20 0.00 26.37 26.84 0.00 0.00 35.63 25.20 34.17 0.00 30.09 25.48 29.66 0.00 29.38 25.21 29.43 0.00 27.58 25.65
Y.pseudotb 0.00 0.00 0.00 25.30 0.00 0.00 0.00 26.03 0.00 0.00 0.00 28.56 0.00 0.00 0.00 26.86 0.00 0.00 0.00 25.25 0.00 0.00 0.00 25.23 0.00 0.00 0.00 25.27 0.00 0.00 0.00 25.64
Yp A1122 13.62 0.00 12.53 0.00 14.43 0.00 13.82 33.42 17.96 0.00 16.44 0.00 18.42 0.00 16.55 29.85 13.72 0.00 12.06 0.00 14.62 0.00 13.72 28.48 14.10 0.00 12.54 0.00 14.77 0.00 14.06 31.22
PC-2 28.64 29.39 26.66 25.30 28.84 25.13 32.72 25.89 21.95 21.44 20.17 0.00 22.73 20.00 24.38 27.43 30.53 31.63 27.99 25.20 33.01 39.06 29.25 25.52 28.53 29.14 26.03 25.18 28.33 32.28 24.74 25.62
NTC 0.00 0.00 0.00 25.28 0.00 0.00 0.00 26.18 0.00 0.00 0.00 28.93 0.00 0.00 0.00 26.96 0.00 0.00 0.00 25.21 0.00 0.00 0.00 25.68 0.00 0.00 0.00 25.25 0.00 0.00 0.00 25.61
Usage: Assay
Curve Analysis: Primary
Threshold Setting: Manual
Manual Threshold Fluorescence Units: 15.0
Auto Min Cycle: 5
Auto Max Cycle: 10
Valid Min Cycle: 3
Valid Max. Cycle: 60
Background subtraction: ON
Boxcar Avg. Cycles: 0
Background Min. Cycle: 5
Background Max. Cycle: 40
Primary fluorescence curves that cross the threshold will be recorded as POS and the cycle
when the sample crossed the threshold will be recorded in the Results Table.
SMARTCYCLER
A qPCR master mix (23 µl) was prepared with species specific primers and probes targeting
virulence plasmids associated with pathogenic Yersinia. A chromosomal target for Y. pestis was
also included in the multiplex qPCR. OmniMix HS beads (Cepheid, Sunnyvale, CA) was used to
complete the master mix. 2 µl of prepared DNA template was used in the final reaction.
Parameters: Dye set--FTTC25, 2-step PCR protocol with initial activation hold of 60 sec at
94°C, followed by 45 cycles of 10 sec at 94°C, (optics off) then 45 sec at 60°C, (optics on).
Interpretation Settings:
On SmartCycler Instrument set the following Analysis Settings for FAM, TET, Texas Red and Cy5
channels. Update analysis settings if they are changed before recording results.
•Amedeo Amedei, Elena Niccolai, Luigi Marino, Mario M. D’Elios, Role of immune response in
Yersinia pestis infection, J Infect Dev Ctries 2011; 5(9):628-639.
•AOAC / OMA Program Manual, May 2005
•Robert D. Perry and J. D. Fetherstone, Yersinia pestis—Etiologic Agent of Plague, Clinical
Microbiology Reviews, Vol. 10, No. 1, Jan. 1997, p. 35–66
•Validation Guidelines for FERN Chemical, Microbiological and Radiological, FERN-ADM.0008.00-
06/22/2010
ABI7500:
PCR master mix (28 µl/reaction) was prepared with species specific primers/probes,
Invitrogen Express qPCR Universal and TaqMan IPC (Life Technologies). 2 µl of template DNA
was used per reaction.
Y. pestis qPCR Master Mix Composition
Master Mix Component
Volume (µl)
per 30µl rxn
Molecular Grade Water 3.49
Express qPCR Universal 15
ATC Primer/Probe Mix1 5.85
IPC Primer/Probe Mix 3
IPC DNA 0.6
ROX 0.06
1This contains Yp 7500 P/P (primers/probes
as described in the SmartCycler Protocol)
Sample homogenate/enrichment: Sample cannot be
ruled out for presence of Yersinia pestis if Yp Chrom
The reduced number of samples testing positive for Y. pestis by culture and qPCR at low spike
levels 1.5 and 1.3 cfu/g, for hot dog and baby food respectively, is most probably due to lack
of target present in the samples which tested negative, rather than a lack of tet sensitivity.
During the sample processing, initially, only one quarter of the original homogenized sample is
spun down and after this, only 1 mL of the pellet suspension, hydrated in 5 mL saline, is
transferred for enrichment, while 4 mL or 80% of the suspension remains untested. Overall the
data indicates that in the detection of Y. pestis in foods with high levels of naturally occurring
competitive flora, where spike levels vary from 3.0 – 390 cfu/g, qPCR detection was
significantly better comparative to the culture method.
Significance:
Rapid screening of food enrichments by qPCR can provide vital information on the presence of
Y. pestis, especially if background microflora adversely affects cultural isolation and recovery.
DISCLAIMER
Any opinions, findings, conclusions, or recommendations expressed in this publication are those
of the author(s) and do not necessarily reflect the view(s) of the U. S. Department of
Agriculture and U.S. Food and Drug Administration.
This material is based upon work supported by the U. S. Department of Agriculture, Food
Safety and Inspection Service, Food Emergency Response Network Cooperative Agreement No
FSIS-C-03-2014 and FDA State Partnership Funding Program.
Instruments: Cepheid - SmartCycler® II and Applied Biosystems® 7500 Fast Dx)
Tested Foods: Hot dog, Meat-based baby food, Ground beef - frozen patties.
Y. pestis strains: BT07226001; BT08275005; BT07310003 (clinical isolates) and A1122.
Culture procedure: A 1:5 dilution was prepared by placing 25g or mL of the sample into a
sterile plastic dual chamber, filtered stomacher bag and adding the appropriate amount of
Buffered Peptone Water. Homogenization was done in a Stomacher for 2 minutes at 200 rpm.
Then 25 mL of the homogenate was centrifuged at 10,000 x g for 15 min at room temperature.
Supernatant was discarded and the pellet was re-suspended in 5mL of 0.85% saline, 1 mL of the
pellet suspension was added to 9 mL of BMW Broth. Enrichments were incubated for 24 hours at
28° ± 2°C. After this all samples were plated for isolation onto selective media including CIN
and Yp CHROM agars. Picks of at least 2 suspect colonies from CIN and Yp CHROM agar were
transferred onto SBA for preliminary identification using gram stain, catalase, oxidase, LFD
immunoassay and qPCR.
Fig. 1 72 hours old Y. pestis culture on CIN agar
(left) and on R & F® Y. pestis Chromogenic Plating
Medium (right).
Fig. 2 48 hours old culture of Y. pestis enrichment
streaked for isolation on CIN agar (left) and on R & F®
Y. pestis Chromogenic Plating Medium (right).
Dye Name Target
FAM Yp Chromosomal Target
TET lcrV plasmid target
TxRed pla plasmid target
Cy5 Internal Control Target
Fig. 3 SC II amplification plot of screening Meat based baby food
(24 hours enrichment) at 0; 1.3 and 13.0 cfu/g Y. pestis load.
In the survey portion of the study, average qPCR detection rates of Y. pestis were 100%, 80%,
50% and 33% from different sources of deli meats, ground beef, ground turkey and raw chicken,
respectively, while cultural recovery rates were 55%, 0%, 0% and 5%, respectively. We did not
observe any false positive reactions in the non-spiked survey samples tested.
Parameters: 2-stage PCR protocol with initial activation hold of 60 sec at 94°C, followed by 45
cycles of 10 sec at 94°C,then 45 sec at 60°C.
Dye Name Target
FAM Yp Chromosomal Target
TAMRA lcrV plasmid target
Cy5 pla plasmid target
VIC Internal Control Target
Fig. 4 ABI 7500 amplification plot of screening Frozen Ground Beef
patties (24 hours enrichment) at 0; 100; 340 cfu/g Y. pestis load
Table 1. Screening results summary table (ct values) of samples surveyed through the course of the study.
target (FAM) crosses threshold (Ct value displayed) with or without the lcrV, pla or IPC targets.