TaqMan Pathogen Detection Kitstools.thermofisher.com/content/sfs/brochures/cms_042473.pdfIntroduction TaqMan® Pathogen Detection Kits are part of a systems approach that helps users

Introduction

TaqMan® Pathogen Detection Kits are part of a systems approach that helps users 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 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

TaqMan® Pathogen Detection Kits

WHITe paper TaqMan® Pathogen Detection Kits — Validation Reference

The protocol consists of five simple steps (Fig. 1):

• Pre-enrichment. In this step, a homogenized food sample (and 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.

Salmonella enterica

Escherichia coli O157:H7

Listeria monocytogenes

Campylobacter jejuni

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

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.

conclusionBased 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.

• 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.

Figure 1. Five-step standardized protocol used in all TaqMan® Pathogen Detection Kits.

Sample Perform Pre-Enrichment

Prepare DNA

Prepare PCR

Perform PCR

View Results

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

* An 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”.

Table 1. Salmonella enTerICa SpeCIFICITy

Inclusivity 100% (51/51)

Exclusivity 100% (25/25)

False Negative Rate 0%

False Positive Rate 0%

Test Efficiency 100%

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.

specificityThe 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.

sensitivityThe 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

5 g of each of 13 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 2.

The PCR results were culture confirmed. The inoculation level at the 10-8 dilution was 6 cells of S. enterica per 5 g of matrix.

Repeatability and ReproducibilityTo test the repeatability of the TaqMan® Salmonella enterica Detection Kit, 10-3 and 10-6 dilutions of the S. enterica 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 3. The assay exhibited a high degree of both repeatability and reproducibility. The relative standard deviation within a test did not exceed 4%.

Table 3. Salmonella enTerICa repeaTabIlITy and reprodUCIbIlITy

10-3 DIlutIon 10-6 DIlutIonS. 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

different E. coli O157:H7 strains ranging from 10-4 to 10-9 were added to 5 g of each of 13 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 6.

The PCR results were culture confirmed. The inoculation level at the 10-9 dilution was 1.5 cells of E. coli O157:H7 per 5 g of matrix.

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

Table 5. eSCHerICHIa ColI o157:H7 SpeCIFICITy






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.

specificityThe 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.

sensitivityThe 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

Repeatability and ReproducibilityTo test the repeatability of the TaqMan® Escherichia coli O157:H7 Detection Kit, 10-3 and 10-6 dilutions of the E. coli O157:H7 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 7. The assay exhibited a high degree of both repeatability and reproducibility. The relative standard deviation within a test did not exceed 4%.

Table 8. STraInS USed In e. ColI o157:H7 SpeCIFICITy TeSTIng

InclusIvIty testIng (+) exclusIvIty testIng (-) exclusIvIty testIng (-)


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

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


Table 9. lISTerIa monoCyTogeneS SpeCIFICITy






Table 10. lISTerIa monoCyTogeneS SenSITIvITy In mIxed CUlTUre

L. monocytogenes mix dilutions 10-4 10-5 10-6 10-7 10-8

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).

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.

taqman® Listeria monocytogenes Detection Kit Listeria monocytogenes, one of six species in the genus Listeria, causes Listeriosis. Listeria mainly poses a severe risk to pregnant women, potentially causing spontaneous abortion or stillbirth. Normally, Listeria is transferred to humans through raw milk, soft-ripened cheeses, raw vegetables, poultry, raw meats, and raw or smoked fish. Listeria can grow at temperatures as low as 3˚C in refrigerated foods.

specificityThe TaqMan® Listeria monocytogenes Detection Kit identifies all serotypes 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 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.

sensitivityThe 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.

For the analysis of spiked food samples, 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.

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.

Repeatability and ReproducibilityTo 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%.

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


Table 13. STraInS USed In lISTerIa monoCyTogeneS SpeCIFICITy TeSTIng

InclusIvIty testIng (+) exclusIvIty testIng (-) exclusIvIty testIng (-)


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

Repeatability and ReproducibilityTo test the repeatability of the TaqMan® Campylobacter jejuni Detection Kit, 10-3 and 10-6 dilutions of the C. jejuni 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 17. The assay exhibited a high degree of both repeatability and repro-ducibility. The relative standard deviation within a test did not exceed 2%.

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).

Table 14. CampylobaCTer SpeCIFICITy

Campylobacter jejuni Campylobacter coli

Inclusivity 100% (19/19) 100%(8/8)





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.

specificityThe 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.

sensitivityThe 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.

For the analysis of spiked food samples, 100-µl aliquots of dilutions of the C. jejuni mix ranging from 10-4 to 10-9 were added to 5 g of each of 8 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 16.

Real-Time PCR identified C. jejuni 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


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Table 18. STraInS USed In CampylobaCTer jejUnI and CampylobaCTer ColI TeSTIng

InclusIvIty testIng (+) InclusIvIty testIng (+) exclusIvIty testIng (-)


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

For Research Use Only. Not for use in diagnostic procedures.

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©2008 Applied Biosystems. All rights reserved. Applied Biosystems, AB (Design), and PrepMan are registered trademarks and Applera and RapidFinder are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc.

Printed in the USA. 02/2008 Publication 116WP01-02

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TaqMan Pathogen Detection Kitstools.thermofisher.com/content/sfs/brochures/cms_042473.pdfIntroduction TaqMan® Pathogen Detection Kits are part of a systems approach that helps users