White Paper TaqMan® Pathogen Detection Kits — Validation Reference

TaqMan Pathogen Detection Kits

®

Salmonella enterica

Escherichia coli O157:H7

Listeria monocytogenes

Campylobacter jejuni

Introduction
TaqMan® Pathogen Detection Kits are
part of a systems approach that helps
user to detect food pathogens both
rapidly and accurately. The kits use
molecular methods (i.e., Real-Time PCR)
to simplify handling and to reduce the
time-to-result. The kits have undergone
rigorous internal testing and extended
external validation as described herein
by an independent service lab. This
document summarizes the protocol and
results of the external validation (except
where noted).
The following four kits were validated:
• TaqMan® Salmonella enterica
Detection Kit
• TaqMan® Escherichia coli O157:H7
Detection Kit
• TaqMan® Listeria monocytogenes
Detection Kit
• TaqMan® Campylobacter jejuni
Detection Kit
“Validations” shall mean testing was
performed to quantify the specificity
(both inclusivity and exclusivity),
sensitivity (in pure sample dilutions
as well as spiked matrices),
and repeatability and reproducibility
of each kit.
Standardized Protocol
All TaqMan Pathogen Detection Kits use
® homogenized food sample (and
a uniform protocol to facilitate simplified
workflows and generate consistent
results. Both the PCR reaction and the
PCR run protocol are standardized so
that multiple pathogens can be screened
on the same plate in the same PCR run.
Each kit is designed for ease-of-use, with
color-coded caps and a software-guided
workflow. The kits include reagents for
preparation and amplification of up to
100 samples, including:
• Environmental master mix (EMM,
a reagent common for all pathogen
detection assays. It contains the
polymerase enzyme that initiates
PCR in the presence of the necessary
primers and DNA template.)
• Target assay mix (TAM, a target-
specific reagent for each pathogen
detection assay. It contains specific
primers and probes for the target.)
• Negative control
The protocol consists of five simple
steps (Fig. 1):
• Pre-enrichment. In this step, a
any viable food-borne bacteria) are
incubated in an enrichment medium
where the bacteria are allowed to
multiply to detectable levels.
• DNA preparation. After pre-
enrichment, the bacterial cells are
separated from the medium and
mixed with a sample preparation
reagent (i.e., PrepMan® Ultra) to lyse
the cells and release their DNA. When
necessary, additional purification steps
may be performed to remove PCR
inhibitors from the reaction.
• PCR preparation. Each PCR reaction
(containing EMM, TAM, and an aliquot
of the unknown DNA sample) is mixed
on the plate.
• PCR performance. The plate is then
run in an Applied Biosystems Real-
Time PCR System to amplify the
target DNA and plot the results.
 Sample Perform
Pre-Enrichment
Figure 1. Five-step standardized protocol used in all TaqMan ® Pathogen Detection Kits.
• Analysis of the results. The
amplification plots are analyzed
either manually or with Applied
Biosystem RapidFinder™ Software.
The internal positive control (IPC)
verifies successful amplification
in the case of a negative result
(inhibition control). The absence of
signal from the negative control rules
out contamination or non-specific
amplification.
Real-Time PCR
Real-time PCR monitors the progress
of the amplification reaction as it occurs
(i.e., in real time). Data are collected
throughout the process, rather than only
at the end. In Real-Time PCR, reactions
are characterized by the point in time
during cycling when amplification of
Figure 2. Campylobacter jejuni specificity testing amplification plot.
Prepare Prepare
DNA PCR
a target is first detected. Specifically,
the threshold cycle (CT) is the fractional
cycle number at which amplification
of the target exceeds the threshold of
detection. In other words, it is the point
at which the reaction returns a positive
result. In general, the higher the CT, the
smaller the number of copies of the
target in the original sample (i.e., the
more amplification cycles required for
the target to be detectable).
Fig. 2 shows a typical amplification
plot, derived in this case from the
Campylobacter jejuni specificity
experiments. The green line represents
the threshold of detection. The red
arrows indicate the CT for the C. jejuni
and internal positive control (IPC),
respectively. The amplification curves of
Perform View Results
PCR
non-target species produce a negative
signal (no amplification) and did not
reach the threshold level.
Pre-Enrichment
Pre-enrichment is the first step in using
the TaqMan® Pathogen Detection Kits.
Pre-enrichment consists of growing
the specific pathogen from a selected
sample. Most standard pre-enrichment
procedures work with the kits and have
been validated in internal testing.
Conclusion
Based on the testing summarized on
the following pages, TaqMan® Pathogen
Detection Kits will help users to identify
food pathogens both rapidly and
accurately using an efficient, easy-to-use
systems approach. In external validation
testing, all four kits had a high degree
of sensitivity and specificity for their
target organisms, both in culture and
in spiked food samples. All four kits
exhibited a high degree of repeatability
and reproducibility.
In combination with Applied Biosystems
Real-Time PCR Systems such as the
7300 and 7500 Systems, RapidFinder
software, and PrepMan® Ultra Sample
Preparation Reagent, TaqMan® Pathogen
Detection Kits offer fully-integrated,
single-vendor solutions that provide
superior performance, reliability, and
ease-of-use.
TaqMan® Salmonella enterica
Detection Kit
More than 2,400 Salmonella serotypes
have been identified, all of which are
potentially pathogenic. S. enterica is a
frequently reported cause of food-borne
illness. Outbreaks have been associated
with raw meats and poultry, eggs,
milk and dairy products, seafood,
coconut sauces, salad dressings,
cocoa, and chocolate.
Specificity
The TaqMan® Salmonella enterica
Detection Kit identifies all subspecies
of S. enterica. The specificity of the
assay has been validated in extensive
inclusivity and exclusivity testing, as
shown in Table 1. The kit detected
100% (51/51) of the S. enterica strains
tested. None of 25 non-S. enterica
strains (see Table 4 for a complete list)
reacted positively. No false negatives
or false positives were observed during
specificity testing.
Sensitivity
The sensitivity of the TaqMan®
Salmonella enterica Detection Kit was
tested both in culture and in spiked
food samples in two separate phases of
testing. The first phase was an in-house
study in which 25-gram samples of 4
different food types (beef 7% fat, beef
20% fat, eggs, and chocolate) were
spiked with 1-3 colony forming units
(CFU). Enrichment broth was added to
bring the total weight to 250 g. After
incubation for 16 hours, Real-Time PCR
was performed. Each experiment was
performed in triplicate. All 4 food types
yielded a positive result, which was
culture confirmed, demonstrating a
sensitivity level of 1 CFU in 25 g of
food after enrichment.
In the second phase, a collaborative
study was conducted to challenge the
protocol by testing a broader range
of food types. In this testing, 100-µl
aliquots of dilutions of a mix of 20
different Salmonella enterica strains
ranging from 10-4 to 10-8 were added to
Table 1. Salmonella enterica specificity
Inclusivity 100% (51/51)
Exclusivity 100% (25/25)
False Negative Rate 0%
False Positive Rate 0%
Test Efficiency 100%
Table 2. Salmonella enterica sensitivity in spiked matrices
Real-Time PCR: Mean CT
Food* 10-7 dilution 10-8 dilution
Beef 17.1 32.1
Pork 18.5 18.8
Chicken 25.2 30.7
Maize (Corn) Oil 16.0 17.4
Butter 19.9 33.0
Sugar 15.8 31.6
Eggs 15.2 32.8
Nuts 18.2 33.9
Corn (Maize) Starch 16.7 18.3
Milk 18.1 22.6
Cheese 19.3 35.2
Chocolate Cream 19.6 39.2
Soy Lecithin 16.8 16.9
*A n additional food type tested was cocoa powder. None of the cocoa samples tested showed
amplification following the standard protocol due to colored compounds transferred into the PCR.
Additional purification is required before running cocoa powder and similar food types as described
in the PrepMan Ultra protocol on page 11-14 “Additional Methods for Separating Fine Particulates”.
5 g of each of 13 different foods Repeatability and Reproducibility
commonly contaminated with the To test the repeatability of the TaqMan®
bacterium. Each experiment was Salmonella enterica Detection Kit, 10-3
performed in triplicate. Enrichment broth and 10-6 dilutions of the S. enterica mix
was added to bring the total weight to lysate were amplified by Real-Time PCR
50 g. After incubation, Real-Time PCR 12 times each. To test the reproducibility
was performed. The results are shown of the kit, the assay was performed at
in Table 2. weekly intervals with three different
operators. The results are shown in
The PCR results were culture confirmed.
Table 3. The assay exhibited a high degree
The inoculation level at the 10-8 dilution
of both repeatability and reproducibility.
was 6 cells of S. enterica per 5 g
The relative standard deviation within a
of matrix.
test did not exceed 4%.
 Table 3. Salmonella enterica repeatability and reproducibility

10 -3 Dilution 10 -6 Dilution
S. enterica mix Mean CT SD %RSD Mean CT SD %RSD

Test 1 24.4 0.08 0.33 33.0 0.51 1.58

Test 2 26.7 0.24 0.92 34.6 0.38 1.11

Test 3 24.3 0.23 0.93 35.2 1.15 3.26

SD, standard deviation; %RSD, percent relative standard deviation.

Table 4. Strains Used in Salmonella enterica Specificity Testing

Inclusivity Testing (+) Inclusivity Testing (+) Exclusivity Testing (-)

Organism Strain/Source Organism Strain/Source Organism Strain/Source

Salmonella abaetetuba ATCC 35640 Salmonella heidelberg ATCC 8326 Bacillus cereus ATCC 10876

Salmonella abony NTCT 6017 Salmonella hemo 2/CI Bacillus subtilis ATCC 6051

Salmonella abortus-equi SL5414 Salmonella infantis 6064 Campylobacter jejuni ATCC 29428

Salmonella abortus-ovis 1132/95 Salmonella isangi 1066/94 Citrobacter freundii 6879

Salmonella adeleide 859/93 Salmonella kunzendorf ATCC 12011 Clostridium perfringens ATCC 12915

Salmonella agona SARB1 Salmonella miami SARB28 Enterobacter aerogenes Q87

Salmonella anatum (Group E) ATCC 9270 Salmonella montgomery 457/89 Enterobacter sakazaki ATCC 51329

Salmonella anatum 10/I2 Salmonella naestyed 1131/95 Enterococcus faecalis ATCC 29212

Salmonella arizonae ATCC 13314 Salmonella newport ATCC 27869 Escherichia coli O157 43888

Salmonella austin SL5747 Salmonella nienstedten 1018/94 Klebsiella oxytoca ATCC 43165

Salmonella boccker 1012/94 Salmonella panama ATCC 7378 Lactobacillus bulgaris ATCC 11842

Salmonella borum 1016/94 Salmonella panama 10/I3 Listeria ivanovii ATCC 19119

Salmonella bovismorbificans SL5747 Salmonella paratyphi (Group A) ATCC 9150 Listeria monocytogenes ATCC 7644

Salmonella carrau 1011/94 Salmonella poona NTCT 4840 Pseudomonas aeruginosa ATCC 27853

Salmonella chandans 456/89 Salmonella tallahassee ATCC 12002 Pseudomonas aeruginosa ATCC 17423

Salmonella choleraesuis ATCC 10708 Salmonella typhi 9/H9 Shigella Sfla 395

Salmonella choleraesius ATCC 7001 Salmonella typhimurium 6765 Shigella SFL 153

Salmonella decatur SARB8 Salmonella typhimurium ser. typhimurium Shigella dysenteriae ATCC 13313

Salmonella dublin ATCC 15480 Salmonella typhimurium 13311 Shigella dysenteriae ESCL7-JHH

Salmonella eimsbutter 1017/94 Salmonella typhimurium 14028 Staphylococcus aureus ATCC 43300

Salmonella enterica 9/B6 Salmonella typhimurium 25241 Staphylococcus aureus aureus PE491

Salmonella enterica I ser. Infantus Salmonella typhimurium ISS 58/3 Streptococcus faecalis ATCC 9790
copenhagen

Salmonella eneritidis (Group D) ATCC 13076 Salmonella vellore (Group B) ATCC 15611 Vibrio cholerae O36

Salmonella give 458/89 Salmonella ser. 18:Z4Z32 11594 Yersinia enterocolitica ATCC 9610

Salmonella hadar PE 55 Salmonella MF350 Salmonella bongori ATCC 43975

Salmonella havana SL5749

TaqMan® Escherichia coli O157:
H7 Detection Kit
E. coli O157:H7, one of over 700
reported pathogenic serotypes of E. coli,
is the causative agent of hemorrhagic
colitis. Outbreaks have been associated
with undercooked meat, unpasteurized
milk or juice, lettuce, game meat, and
raw milk.
Specificity
The TaqMan® Escherichia coli O157:H7
Detection Kit identifies serotypes O157:
H7 and O55:H7. It also detects some
pathogenic, non-motile strains of E. coli
O157:NM. The specificity of the kit has
been validated in extensive inclusivity
and exclusivity testing, as shown in
Table 5. The kit detected 100% (16/16) of
the E. coli O157:H7 strains tested. None
of 31 other E. coli strains or 21 strains
from other bacterial species (see Table 8
for a complete list) reacted positively. No
false negatives or false positives were
observed during specificity testing.
Sensitivity
The sensitivity of the TaqMan®
Escherichia coli O157:H7 Detection
Kit was tested both in culture and in
spiked food samples. in two separate
phases of testing. The first phase was
an in-house study in which 25-gram
samples of 4 different food types (apple
juice, ground beef, whole milk, and
alfalfa sprouts) were spiked with 1-3
CFU. Enrichment broth was added to
bring the total weight to 250 g. After
incubation for 16 hours, Real-Time PCR
was performed. Each experiment was
performed in triplicate. All 4 food types
yielded a positive result, which was
culture confirmed, demonstrating a
sensitivity level of 1 CFU in 25 g of food
after enrichment.
In the second phase, a collaborative
study was conducted to challenge the
protocol by testing a broader range of
food types. In this testing, 100-µl
aliquots of dilutions of a mix of 3
Table 5. Escherichia coli O157:H7 specificity
Inclusivity 100% (16/16)
Exclusivity 100% (52/52)
False Negative Rate 0%
False Positive Rate 0%
Test Efficiency 100%
Table 6. Escherichia coli O157:H7 detection in spiked matrices
Real-Time PCR: Mean CT
Food 10-8 dilution 10-9 dilution
Beef 31.7 33.1
Turkey 33.0 32.9
Salami 20.1 34.6
Ham Sandwich 31.1 29.6
Salad 30.4 34.3
Sprouts 29.0 35.4
Potato 21.5 35.0
Cantaloupe 21.7 34.1
Apple Juice 17.7 19.3
Milk 19.7 36.5
Hard Cheese 19.8 33.2
Semi-Soft Cheese 29.1 31.2
Mayonnaise 16.4 28.5
different E. coli O157:H7 strains ranging Repeatability and Reproducibility
from 10-4 to 10-9 were added to 5 g of To test the repeatability of the TaqMan®
each of 13 different foods commonly Escherichia coli O157:H7 Detection Kit,
contaminated with the bacterium. Each 10-3 and 10-6 dilutions of the E. coli O157:
experiment was performed in triplicate. H7 mix lysate were amplified by Real-
Enrichment broth was added to bring the Time PCR 12 times each. To test the
total weight to 50 g. After incubation, reproducibility of the kit, the assay
Real-Time PCR was performed. The was performed at weekly intervals
results are shown in Table 6. with three different operators. The
results are shown in Table 7. The
The PCR results were culture confirmed.
assay exhibited a high degree of
The inoculation level at the 10-9 dilution
both repeatability and reproducibility.
was 1.5 cells of E. coli O157:H7 per 5 g
The relative standard deviation within
of matrix.
a test did not exceed 4%.
 Table 7. Escherichia coli O157:H7 repeatability and reproducibility

E. coli 10 -3 Dilution 10 -6 Dilution

O157:H7 mix Mean CT SD %RSD Mean CT SD %RSD

Test 1 22.8 0.15 0.64 33.0 0.50 1.50

Test 2 23.0 0.34 1.46 32.8 1.12 3.40

Test 3 20.2 0.13 0.64 31.6 0.50 1.58

SD, standard deviation; %RSD, percent relative standard deviation.

Table 8. Strains Used in E. coli O157:H7 Specificity Testing

Inclusivity Testing (+) Exclusivity Testing (-) Exclusivity Testing (-)

Organism Strain/Source Organism Strain/Source Organism Strain/Source

Escherichia coli O157:H7 ATCC 43890 Escherichia coli 5190 Escherichia coli O26:H32 ECRC#88-0430

Escherichia coli O157:H7 380-95 Escherichia coli 4157 Escherichia coli O55:H9 ECRC#86-0680

Escherichia coli O157:H7 C7927 Escherichia coli 8739 Escherichia coli O154:H25 94.0726

Escherichia coli O157:H7 C9490 Escherichia coli 10536 Escherichia coli O156:H8 93.0541

Escherichia coli O157:H7 96A5997 Escherichia coli 11229 Bacillus cereus ATCC 10876

Escherichia coli O157:H7 96A12165 Escherichia coli 11775 Bacillus cereus PE1123

Escherichia coli O157:H7 96A12374 Escherichia coli 13706 Bacillus subtilis ATCC 11838

Escherichia coli O157:H7 96A12814 Escherichia coli 13762 Citrobacter freundii 6879

Escherichia coli O157:H7 43888 Escherichia coli 14948 Clostridium perfringens ATCC 12915

Escherichia coli O157:H7 700728 Escherichia coli 25922 Enterobacter sakazaki ATCC 51329

Escherichia coli O157:H7 NTCT 12900 Escherichia coli 33605 Enterococcus faecalis ATCC 29212

Escherichia coli O157:NM MF13180-25 Escherichia coli 35218 Listeria monocytogenes 7057

Escherichia coli O55:H7 1880 Escherichia coli 35421 Listeria monocytogenes ATCC 7644

Escherichia coli O55:H7 10591-12-74 Escherichia coli 51446 Pseudomonas aeruginosa ATCC 27853

Escherichia coli O55:H7 4694-9-75 Escherichia coli 51755 Pseudomonas aeruginosa ATCC 17423

Escherichia coli O55:H7 2022-7-76 Escherichia coli 51813 Salmonella enteritidis ATCC 13076

Escherichia coli 9637 Salmonella enterica I ser. Infantus

Escherichia coli 23848 Shigella Sfla 395

Escherichia coli 33876 Shigella SFL 153

Escherichia coli O26:H11 3359-70 Shigella dysenteriae 88.0607

Escherichia coli O145:NM 88.0963 Shigella dysenteriae ATCC 13313

Escherichia coli O78:K80:H12 43896 Staphylococcus aureus PE491

Escherichia coli O103:H2 87.1368 Streptococcus faecalis ATCC 9790

Escherichia coli O5:NM 85.0587 Vibrio cholerae O36

Escherichia coli O137:H41 88A5333 Yersinia enterocolitica ATCC 9610

Escherichia coli O48:H21 94A9057

Escherichia coli O28:H35 96A11759

TaqMan® Listeria
Table 9. Listeria monocytogenes specificity
monocytogenes Detection Kit
Listeria monocytogenes, one of Inclusivity 100% (29/29)
six species in the genus Listeria,
causes Listeriosis. Listeria mainly Exclusivity 100% (37/37)
poses a severe risk to pregnant
False Negative Rate 0%
women, potentially causing spontaneous
abortion or stillbirth. Normally, Listeria is False Positive Rate 0%
transferred to humans through raw milk,
soft-ripened cheeses, raw vegetables, Test Efficiency 100%
poultry, raw meats, and raw or smoked
fish. Listeria can grow at temperatures
as low as 3˚C in refrigerated foods.
Table 10. Listeria monocytogenes sensitivity in mixed culture
Specificity
The TaqMan® Listeria monocytogenes L. monocytogenes
Detection Kit identifies all serotypes mix dilutions 10-4 10-5 10-6 10-7 10-8

of the species L. monocytogenes.

The specificity of the assay has been
validated in extensive inclusivity and
exclusivity testing, as shown in Table 9.
The kit detected 100% (29/29) of the
Mean CT value 30.1 33.3 37.2 40.3 36.3
Mean CFU UC UC 22 3 0
CFU, colony-forming units; UC, uncountable (too many colonies to count).

L. monocytogenes strains tested.

None of 15 strains from other Listeria
species or 22 strains from non-Listeria
species (see Table 13 for a complete list)
reacted positively. No false negatives
or false positives were observed during
specificity testing.
Sensitivity
The sensitivity of the TaqMan®
Listeria monocytogenes Detection
Kit was tested both in culture and in
spiked food samples. Ten different L.
monocytogenes strains were combined
and the mixed culture was serially
diluted in 10-fold increments in 0.9%
NaCl. Dilutions of the mixed culture from
10-4 to 10-9 were analyzed in triplicate
using both Real-Time PCR and traditional
plate counts. The results are shown
in Table 10. Real-Time PCR positively
detected L. monocytogenes down to
the 10-8 dilution, equivalent to <1 CFU.
Table 11. Listeria monocytogenes sensitivity in spiked matrices
Real-Time PCR: Mean CT Sensitivity
Food 10-8 dilution 10-9 dilution CFU/25 g
Ground Beef 35.2 33.2 <1.0
Chicken 35.8 37.4 <1.0
Frankfurters 39.2 36.2 <1.0
Sliced Ham 36.8 36.8 <1.0
Shrimp 37.0 36.0 <1.0
Lettuce 35.4 37.0 <1.0
Spinach 38.9 37.8 <1.0
Green Beans 40.1 34.0 <1.0
Milk 33.5 34.6 <1.0
Hard Cheese 37.8 41.5 <1.0
Brie Cheese 44.1 36.4 <1.0
Yogurt 37.7 Undetermined 1.5
Egg 34.5 38.3 <1.0
Mayonnaise 24.1 35.9 <1.0

Undetermined, amplification did not exceed the threshold level.

 Table 12. Listeria monocytogenes repeatability and reproducibility

L. monocytogenes 10 -3 Dilution 10 -6 Dilution

mix Mean CT SD %RSD Mean CT SD %RSD

Test 1 26.0 0.29 1.11 35.2 1.86 5.28

Test 2 23.3 0.47 2.02 36.3 1.93 5.31

Test 3 21.8 0.52 2.38 33.3 2.25 6.76

SD, standard deviation; %RSD, percent relative standard deviation.

For the analysis of spiked food samples, Repeatability and Reproducibility

100-µl aliquots of dilutions of the
L. monocytogenes mix ranging from 10-4
to 10-9 were added to 5 g of each of 14
different foods commonly contaminated
with the bacterium. Each experiment
was performed in triplicate. Enrichment
broth was added to bring the total
weight to 50 g. After incubation, Real-
Time PCR was performed. The results
are shown in Table 11.
Real-Time PCR identified L.
monocytogenes positively down to
the 10-9 dilution in all the spiked matrices
except yogurt. At the 10-9 dilution,
the inoculation level was <1 cell of L.
monocytogenes per 5 g of matrix.
To test the repeatability of the TaqMan®
Listeria monocytogenes Detection
Kit, 10-3 and 10-6 dilutions of the L.
monocytogenes mix lysate were
amplified by Real-Time PCR 12 times
each. To test the reproducibility of the
kit, the assay was performed at weekly
intervals with three different operators.
The results are shown in Table 12. The
assay exhibited a high degree of both
repeatability and reproducibility. The
relative standard deviation within a test
did not exceed 7%.

In this experiment, the sensitivity of

the assay was lower in yogurt. Positive
identification occurred at the 10-8, but not
the 10-9 dilution. At the 10-8 dilution, the
inoculation level was 0.3 cells per 5 g
of matrix.
Table 13. Strains Used in Listeria monocytogenes Specificity Testing

Inclusivity Testing (+) Exclusivity Testing (-) Exclusivity Testing (-)

Organism Strain/Source Organism Strain/Source Organism Strain/Source

Listeria monocytogenes LS24 (1b) Bacillus cereus ATCC 10876 Pseudomonas aeruginosa ATCC 17423

Listeria monocytogenes LS113 (1/2a) Bacillus cereus PE1123 Pseudomonas aeruginosa ATCC 27853

Listeria monocytogenes LS80 (1/2b) Bacillus subtilis ATCC 6051 Salmonella enterica I ser. Infantus

Listeria monocytogenes LS115 (1/2c) Campylobacter jejuni ATCC 29428 Shigella dysenteriae ESCL7-JHH

Listeria monocytogenes LS111 (3a) Citrobacter freundii 6879 Shigella dysenteriae ATCC 13313

Listeria monocytogenes LS225 (4a) Clostridium perfringens ATCC 12915 Staphylococcus aureus ATCC 43300

Listeria monocytogenes LS2/ScottA (4b) Enterobacter aerogenes Q87 Staphylococcus aureus aureus Q87

Listeria monocytogenes LS244 (4ab) Enterobacter sakazaki ATCC 51329 Streptococcus faecalis ATCC 9790

Listeria monocytogenes LS21 Enterococcus faecalis ATCC 29212 Vibrio cholerae O36

Listeria monocytogenes Q91 (1) Escherichia coli O157 43888 Yersinia enterocolitica ATCC 9610

Listeria monocytogenes Q98 (1b) Klebsiella oxytoca ATCC 43165

Listeria monocytogenes Q101 (4) Lactobacillus bulgaris ATCC 11842

Listeria monocytogenes Q126 (1) Listeria grayi ATCC 19120

Listeria monocytogenes Q128 (4) Listeria grayi RF4738

Listeria monocytogenes Q158 (4b) Listeria grayi murrayi ATCC 25401

Listeria monocytogenes Q159 (3b) Listeria innocua ATCC 33090

Listeria monocytogenes Q160 (1b) Listeria innocua LS7

Listeria monocytogenes Q161 (1.1/2a) Listeria innocua Q94 (4)

Listeria monocytogenes (1a) Listeria innocua Q94 (4)

Listeria monocytogenes ATCC 7544 Listeria ivanovii ATCC 19119

Listeria monocytogenes ATCC 15313 Listeria murrayi LS39

Listeria monocytogenes ATCC 19111 ser. 1 Listeria seeligeri LS22 (3b)

Listeria monocytogenes ATCC 19112 ser. 2 Listeria seeligeri LS56

Listeria monocytogenes ATCC 19114 ser. 4a Listeria seeligeri ATCC 35967

Listeria monocytogenes ATCC19115 ser. 4b Listeria welshimeri LS128

Listeria monocytogenes ATCC 19113 Listeria welshimeri LS156

Listeria monocytogenes DSM 15675 Listeria welshimeri ATCC 35897

Listeria monocytogenes DSM 12464

Listeria monocytogenes NTCT 10890

TaqMan® Campylobacter jejuni
Detection Kit
Campylobacter jejuni and Campylobacter
coli are the primary cause of bacterial
diarrheal illness in the United States,
causing more cases of disease than
Shigella and Salmonella combined.
Outbreaks of Campylobacteriosis
has been associated with raw chicken,
raw milk, raw clams, and non-
chlorinated water.
Specificity
The TaqMan® Campylobacter jejuni
Detection Kit identifies pathogenic
strains of C. jejuni and C. coli. The
specificity of the assay has been
validated in extensive inclusivity and
exclusivity testing, as shown in Table 14.
The kit detected 100% (19/19) of the
C. jejuni strains and 100% (8/8) of the
C. coli strains tested. None of 10 strains
from other Campylobacter species or 18
strains from unrelated species (see Table
18 for a complete list) reacted positively.
No false negatives or false positives
were observed during specificity testing.
Sensitivity
The sensitivity of the TaqMan®
Campylobacter jejuni Detection Kit was
tested both in culture and in spiked food
samples. Ten different C. jejuni strains
were combined and the mixed culture
was serially diluted in 10-fold increments
in 0.9% NaCl. Dilutions of the mixed
culture from 10-4 to 10-9 were analyzed
in triplicate using both Real-Time PCR
and traditional plate counts. The results
are shown in Table 15. Real-Time PCR
positively detected C. jejuni down to the
10-9 dilution, equivalent to <1 CFU.
Table 14. Campylobacter specificity
Campylobacter jejuni Campylobacter coli
Inclusivity 100% (19/19) 100%(8/8)
Exclusivity 100% (29/29)
False Negative Rate 0%
False Positive Rate 0%
Test Efficiency 100%
Table 15. Campylobacter jejuni sensitivity in pure culture
Campylobacter jejuni
mix dilutions 10-4 10-5 10-6 10-7 10-8 10-9
Mean CT value 29.1 33.4 35.2 37.9 35.5 38.5
Mean CFU UC UC UC 20 2 0
CFU, colony-forming units; UC, uncountable (too many colonies to count).
For the analysis of spiked food samples, Repeatability and Reproducibility
100-µl aliquots of dilutions of the To test the repeatability of the TaqMan®
C. jejuni mix ranging from 10-4 to 10-9 Campylobacter jejuni Detection Kit, 10-3
were added to 5 g of each of 8 different and 10-6 dilutions of the C. jejuni mix
foods commonly contaminated with lysate were amplified by Real-Time PCR
the bacterium. Each experiment was 12 times each. To test the reproducibility
performed in triplicate. Enrichment broth of the kit, the assay was performed at
was added to bring the total weight to weekly intervals with three different
50 g. After incubation, Real-Time PCR operators. The results are shown in
was performed. The results are shown Table 17. The assay exhibited a high
in Table 16. degree of both repeatability and repro-
ducibility. The relative standard deviation
Real-Time PCR identified C. jejuni
within a test did not exceed 2%.
positively down to the 10-9 dilution in
all the spiked matrices except pork and
salad. At the 10-9 dilution, the inoculation
level was 0.2 cells of C. jejuni per 5 g
of matrix.
 Table 16. Campylobacter jejuni sensitivity in spiked matrices

Real-Time PCR: Mean CT Sensitivity

Food 10-7 dilution 10-8 dilution 10-9 dilution CFU/25 g

Ground Beef 35.4 36.2 35.7 1.0

Beef 30.2 32.7 37.5 1.0

Chicken 35.3 37.3 36.1 1.0

Chilled Meal 33.1 36.0 35.7 1.0

Rocket 35.8 35.2 37.9 1.0

Milk 17.9 Undetermined 30.5 1.0

Egg 37.0 36.2 35.3 1.0
Water 18.0 37.3 29.6 1.0

Undetermined, amplification did not exceed the threshold level.

Table 17. Campylobacter jejuni repeatability and reproducibility

10 -3 Dilution 10 -6 Dilution
C. jejuni mix Mean CT SD %RSD Mean CT SD %RSD

Test 1 28.4 0.07 0.24 36.2 0.70 1.94

Test 2 27.6 0.20 0.73 34.0 0.44 1.29

Test 3 27.7 0.07 0.27 33.7 0.38 1.12

SD, standard deviation; %RSD, percent relative standard deviation.

 Table 18. Strains Used in Campylobacter jejuni and Campylobacter coli Testing

Inclusivity Testing (+) Inclusivity Testing (+) Exclusivity Testing (-)

Organism Strain/Source Organism Strain/Source Organism Strain/Source

Campylobacter jejuni subsp. jejuni VPI H840 [CIP] Campylobacter coli LRA 069.05.89 Campylobacter laridis Stanford/BL

Campylobacter jejuni subsp. jejuni AS-83-79 Campylobacter coli Inn 183 Campylobacter laridis 35221

Campylobacter jejuni subsp. jejuni LRA 094.06.89 Campylobacter coli BA 370 Campylobacter fetus subsp. fetus NADL 1083-2255

Campylobacter jejuni C31 Campylobacter coli 33559 Campylobacter fetus subsp. fetus 33561

Campylobacter jejuni ATCC 29428 Campylobacter coli 43475 Campylobacter fetus veneralis 33561

Campylobacter jejuni No. 1 Campylobacter coli 43133 Campylobacter sputorum sputorum 35980

Campylobacter jejuni No. 2 Campylobacter coli 49299 Campylobacter sputorum bubulus 33562

Campylobacter jejuni No.3 Campylobacter coli 43483 Campylobacter sputorum faecalis 33711

Campylobacter jejuni No. 4 Campylobacter concisus 33237

Campylobacter jejuni 63R Campylobacter curves 35224

Campylobacter jejuni 81 Arcobacter cryaeophilus LRA 077.02.87

Campylobacter jejuni 89 Bacillus cereus ATCC 10876

Campylobacter jejuni 194 Enterobacter aerogenes Q87

Campylobacter jejuni ATCC 33560 Enterobacter aerogenes ATCC 13048

Campylobacter jejuni 3135 Escherichia coli O157 43888

Campylobacter jejuni 3138 Heliobacter pylori 87A300

Campylobacter jejuni 33291 Heliobacter pylori RSB6 NCTC

Campylobacter jejuni 49943 Heliobacter pylori TX30A NCTC

Campylobacter jejuni 33560 Listeria monocytogenes 7057

Listeria monocytogenes ATCC 7644

Pseudomonas aeruginosa ATCC 17423

Pseudomonas aeruginosa ATCC 27853

Salmonella enterica I ser. Infantus

Shigella dysenteriae ATCC 13313

Shigella dysenteriae ESCL7-JHH

Staphylococcus aureus ATCC 43300

Staphylococcus aureus aureus PE 491

Streptococcus faecalis ATCC 9790

Vibrio cholerae O36

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