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Junge et al.: Journal of aoaC InternatIonal Vol. 94, no. 6, 2011

FOOD BIOLOGICAL CONTAMINANTS

BIOTECON Diagnostics foodproof ® E. coli O157 Detection  Kit, 5′ Nuclease for E. coli O157 in Combination with  foodproof ShortPrep II Kit

Performance Tested Method SM  100601

Abstract

The method describes the detection of Escherichia coli O157 in food. The method is based on real-time PCR using hydrolysis probes (5′ Nuclease). This advanced PCR method was designed to reduce the time necessary to achieve results from PCR reactions and enable the user to monitor the amplification of the PCR product simultaneously in real time. After DNA isolation using the BIOTECON foodproof ® ShortPrep II Kit designed for the rapid preparation of E. coli O157 DNA for direct use in PCR, the real-time detection of E. coli O157 DNA is carried out using the foodproof E. coli O157 Detection Kit. The kit provides primers and hydrolysis probes for sequence-specific detection, convenient premixed reagents, and controls for reliable interpretation of results. For repeatability studies three different foods (egg salad, large bockwurst/frankfurter, and apple juice) were analyzed, chosen from the 15 food groups recommended by the AOAC Research Institute for E. coli O157 detection. From each food, 20 samples were inoculated with a low level (1–10 CFU/25 g) and 20 samples with a high level (10–50 CFU/25 g) of E. coli O157. Additionally, five nonspiked samples were prepared from each food. Depending on the matrix, the food samples were examined with the test kits and compared with the cultural methods according to the U.S. Food and Drug Administration Bacteriological Analytical Manual or the U.S. Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook.

Participants

Method authorS Benjamin junge, Cordt grönewald, and Kornelia Berghof-jäger

BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam, Germany

Submitted for publication March 25, 2011. The method was independently tested, evaluated, and certified by the AOAC Research Institute as a Performance Tested Method SM . See http://www.aoac.org/testkits/steps.html for information on certification. Corresponding author’s e-mail: bjunge@bc-diagnostics.com DOI: 10.5740/jaoacint.11-097

SubMIttIng CoMpany

BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam, Germany

reVIewerS thomas hammaCK and Yi Chen

U.S. Food and Drug Administration, 5100 Paint Branch Pkwy, College Park, MD 20740

Introduction

Principle

The method describes the detection of E. coli O157 in food. The method is based on real-time PCR. The

foodproof ShortPrep II Kit is used for the isolation of

E. coli O157 DNA from enriched food samples. The

foodproof E. coli O157 Detection Kit detects E. coli O157 specific DNA by means of real-time PCR using 5′ Nuclease- (TaqMan ® )-based instruments.

General Information

Target organisms.—Although most strains of the species Escherichia coli are harmless and live in the intestines of healthy humans and animals, strains of serotype O157 (with a few exceptions) produce a powerful toxin and can cause severe illness. Especially E. coli O157:H7 but also other E. coli O157 serotypes are an emerging cause of foodborne illness. An estimated 73 000 cases of infection

and 61 deaths occur in the United States each year. Infection often leads to bloody diarrhea, and occasionally to kidney failure. Most illness has been associated with eating undercooked, contaminated ground beef (1). The LightCycler foodproof E. coli O157 Detection Kit detects all E. coli of serotype O157 including the O157:H7 serotype. Matrixes.—Three food groups recommended by the AOAC Research Institute for E. coli O157 were tested:

egg salad, large bockwurst/frankfurter, and apple juice. Summary of validated performance claims.—For the repeatability study three different food samples out of the 15 food groups were tested with the foodproof

E. coli O157 Detection Kit, 5′ Nuclease in combination

with the foodproof ShortPrep II Kit. The food samples were tested in correlation with the cultural methods

Junge et al.: Journal of aoaC InternatIonal Vol. 94, no. 6, 2011

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Table 1.

Repeatability study: E. coli O157 strains

and foods

Food

BCD a Strain No.

Serotype

Egg salad

14178

O157:H-

Bockwurst

14226

O157:H7

Apple juice

14244

O157:H7

a BCD = BIOTECON Diagnostics GmbH.

according to U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM) and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS).

Summary of Results

The repeatability study/method comparison study was accomplished with three different food matrixes. The results of the three low and high inoculated foods showed a very high correlation with the PCR method and the cultural FDA-BAM or USDA/FSIS methods. All uninoculated foods were negative. The inclusivity and exclusivity studies gave the expected results on both real-time PCR instruments (LC 480 and Mx3005P). No false positives or false negatives occurred. The specificity was confirmed at

100%.

Materials and Method

Test Kit Information

(a) Kit names.—foodproof E. coli O157 Detection Kit, 5′ Nuclease foodproof ShortPrep II Kit. (b) Cat. Nos.—Cat. No. R 302 10; foodproof E. coli O157 Detection Kit, 5′ Nuclease. Cat. No. S 400 02; foodproof ShortPrep II Kit.

Test Kit Reagents

(a) foodproof E. coli O157 Detection Kit, 5Nuclease.— Reagent 1.—foodproof E. coli O157

Master Mix, ready-to-use primer, and hydroysis probes for E. coli O157 DNA and the E. coli O157-specific internal control (yellow cap). Reagent 2.—foodproof

E. coli O157 Enzyme Solution, contains Taq DNA

polymerase and uracil-DNA glycosylase (heat-labile) for prevention of carryover contamination (red cap). Reagent 3.—foodproof E. coli O157 internal control, contains a stabilized solution of plasmid DNA and a yellow dye for

better visualization (white cap). Reagent 4.—foodproof

E. coli O157 control template, contains a stabilized

solution of plasmid DNA for use as a PCR run positive control (purple cap). Reagent 5.—H 2 O, PCR-grade, contains nuclease-free, PCR-grade H 2 O (colorless cap).

Table 2.

detection kit

Reagents of the foodproof E. coli O157

Component

Volume, mL

foodproof E. coli O157 Master Mix (vial 1)

18.0

foodproof E. coli O157 Enzyme Solution (vial 2)

1.0

foodproof E. coli O157 Internal Control (vial 3)

1.0

Total volume

20.0

(b) foodproof ShortPrep II Kit.—Reagent 1.—

Reaction tubes with 800 µL ready-to-use foodproof ShortPrep II Lysis Reagent. Reagent 2.—Bottle with 20 mL foodproof ShortPrep II Resuspension Reagent.

Additional Supplies and Reagents

(a) Microbiology laboratory equipment.—e.g., Nuclease-

free, aerosol-resistant pipet tips; pipets with disposable,

positive-displacement tips; sterile reaction (Eppendorf) tubes for preparing master mixes and dilutions; LightCycler capillaries, Roche Diagnostics (Mannheim, Germany) Product No. 1 909 339; LightCycler Color Compensation Kit, Roche Diagnostics Product No. 2 158 850; optional:

LightCycler Carousel Centrifuge, Roche Diagnostics Product No. 2 189 682 or 3 030 512; 96-well plates for Mx3005P, e.g., 4titude Cat. No. 4ti-0710/B.

(b) Media and reagents for E. coli O157.—BAM,

Chapter 4a (July 2009) were used.

(c) Media and reagents for E. coli O157.—USDA

FSIS, MLG 5.04 and MLG 5A.01 for meat (January 2008) were used.

(d) Equipment and materials for E. coli O157

BAM assay.—Balance >2 kg with 0.1 g sensitivity; stomacher; incubators, 35 ± 0.5°C and 44 ± 1°C; Petri

dishes 20 × 150 mm; Pasteur pipets; pH test paper, range 6.08.0; screw cap stomacher strainer; and 400 sterile filter bags or equivalent.

(e) Equipment and materials for E. coli O157 EN

ISO 16654 assay.—Autoclave; incubators; water bath; pH analyzer; measuring cylinder; pipets, 25, 10, and 1 mL; tubes, 16 × 125 mm or other appropriate sizes; screw cap stomacher strainer; 400 sterile filter bags or

Table 3.

fluorescence channels

Interpretation of results using two different

E. coli O157 channel FAM

Internal control

Result

channel VIC/HEX

interpretation

Positive

Positive

Positive

Negative

Positive

Negative

Positive

Negative

Positive

Negative

Negative

Invalid

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Junge et al.: Journal of aoaC InternatIonal Vol. 94, no. 6, 2011

Table 4.

microbiologically according to the FDA-BAM or USDA/FSIS methods

Results of the internal comparison study with three food matrixes tested by PCR and

 

Inoculation level (determination via MPN), cells/25 g

Inoculation level,

Cultural

FDA-BAM or

Food

No. of samples

cells/1 g

PCR

confirmation

USDA/FSIS

Egg salad a

20

1.1

0.04

5

5

6

20

11.5

0.46

16

16

15

5

0

0

0

Bockwurst b

20

0.2

0.007

6

6

6

20

5.8

0.23

10

10

10

5

0

0

0

Apple juice a

20

0.4

0.015

7

7

7

20

5.8

0.23

14

14

14

5

0

0

0

a Food matrixes tested according to the FDA-BAM method.

b Food matrix tested according to the USDA/FSIS method.

equivalent; apparatus for immunomagnetic separation, inoculating loops; inoculating needle; microscope slides; needle, scalpels, chisels, knives, scissors, spatulas, forceps, disposable or reusable dishes, pans or trays; Petri dishes, 90 to 140 mm; and vortex mixer.

Apparatus

(a) LightCycler ® 480 real-time PCR system.Roche Diagnostics. The LightCycler 480 real-time PCR system is a high throughput gene quantification or genotyping real-time PCR platform with exchangeable blocks for 96 and 384 samples in multiwell plates. It offers enhanced throughput, compatibility with automation, and maximum flexibility regarding hardware and software. The LightCycler 480 real-time PCR system can complete a 96 well PCR run in 40 min. The LightCycler 480 real-time PCR system is a multiwell-plate based real-time PCR platform that is used for highly accurate qualitative and quantitative detection of nucleic acids and genotyping. Building on the benefits of Roche’s capillary-based LightCycler 1.5 and 2.0 systems, it goes one step further in offering enhanced throughput, compatibility with automation

Table 5.

samples (10–50 CFU/25 g)

Χ 2 values of the high inoculated food

Food

No. of PCR positive/reference method negative (a)

No. of PCR negative/reference method positive (b)

Χ 2

Egg salad

1

0

0

Bockwurst

0

0

0

Apple juice

0

0

0

equipment, and maximum flexibility regarding hardware and software.

(b) Mx3005P QPCR system.Agilent Technologies

(Santa Clara, CA). The Mx3005P system is among the most reliable and trusted QPCR instruments available, with a long record of citation in peer-reviewed journals. Offering unmatched flexibility and reliability, the system is ideal for a wide variety of applications and chemistries, including but not limited to gene expression analysis, microarray data validation, single nucleotide polymorphism genotyping, pathogen detection, DNA methylation analysis, and chromatin immune- precipitation studies. Highly reproducible results are the product of the Mx3005P’s single light source, single detector precision optic scanning design, providing uniform excitation and

detection, coupled with the trusted Peltier-based thermal system, which ensures uniform ramping and thermal accuracy.

(c) Pentium computer with LightCycler and MxPro

software.—For analysis and storage of results.

(d) Bench-top microcentrifuge.—Rotor size to fit

2.0 mL reaction tubes (e.g., Eppendorf Centrifuge

5415C).

(e) Bench-top vortexing unit.—To mix/vortex samples

during foodproof ShortPrep procedure (e.g., Heidolph, REAX 2000, Germany). (f) Heating unit.—e.g., Eppendorf ThermoStat plus 5352 000.0.

Standard Reference Materials

For each food matrix one different E. coli O157 strain was used. Table 1 shows the foods and the applied E. coli O157 strains with their origin and properties.

Junge et al.: Journal of aoaC InternatIonal Vol. 94, no. 6, 2011

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Table 6.

samples (1–10 CFU/25 g)

Χ 2 values of the low inoculated food

Food

No. of PCR positive/reference method negative (a)

No. of PCR negative/reference method positive (b)

Χ 2

Egg salad

0

1

0

Bockwurst

0

0

0

Apple juice

0

0

0

Standard Solutions

(a) Pre-enrichment media according to FDA-BAM

(Feng, P., September 2002, BAM Online, Chapter 4a:

Diarrheagenic E. coli O157).

(b) Pre-enrichment media according to USDA/FSIS

(Microbiology Laboratory Guidebook 5.04 for meat).

General Preparation

The heating unit was turned on and temperature was set to 95100°C. The container was filled with ice for storage of foodproof ShortPrep II kit samples. All sample tubes were labeled for identification of samples. The LightCycler 480 and Mx3005P instruments and attached PCs were turned on.

Sample Preparation

Pre-enrichment.To 25 g of the food sample 225 mL pre-enrichment media was added according to the FDA- BAM and USDA/FSIS instructions.

Analysis

(a) foodproof ShortPrep II kit (Cat. No. S 400 02),

preparation/extraction of DNA from food enrichment.— In order to collect the lysis reagent at the bottom of the

tube, the reaction tube with the ready-to-use reagent was centrifuged at 500 × g for 30–60 s. The enriched culture was shaken gently and allowed to settle for 510 min; 200 µL of the sample (supernatant) was then transferred to the reaction tube containing the ready-to-use lysis reagent. The reaction tube had to be firmly closed. The lysis reagent and sample were mixed by vortexing. The reaction tube was centrifuged in a bench-top microcentrifuge for 5 min at 8000 × g. The supernatant was discarded by pipetting and inactivated appropriately. A 200 µL volume of resuspension reagent was added. The tube was placed in the cell disruption unit and turned on for 8 min for mechanical disruption. The reaction tube was incubated in the heating unit for 10 min at 95100°C. The reaction tube was carefully removed from the heating unit, using forceps, as the tube was hot. The tube was allowed to sit for 1 min, then the sample was mixed for

2 s by vortexing. After centrifugation at 13 000 × g for

1 min at room temperature, the supernatant contained the extracted DNA. The prepared sample was stored at 4°C

if

the PCR followed immediately; otherwise it was stored

at

–20°C.

(b) foodproof E. coli O157 Detection Kit, 5Nuclease

(Cat. No. R 302 10), preparation of the PCR mix.—A

25 µL standard reaction was prepared as described below. Note: Gloves must be used when handling the PCR vessels. The solutions were thawed and, for maximal recovery of contents, vials were briefly centrifuged in

a microcentrifuge before opening. The solutions were

mixed carefully but thoroughly by pipetting up and down. In a reaction tube (0.5–2.0 mL, depending on the number of reactions), the PCR mix was prepared by adding the following components in the order mentioned below. The volumes indicated are based on a single 25 µL standard reaction. The PCR mix was prepared by multiplying the amount in the “Volume” column by the number of reactions to be cycled plus one or two additional reactions to cover pipetting losses (Table 2).

Instructions

(1) Mix carefully but thoroughly by pipetting up and

down. Do not vortex. Pipet 20 µL PCR mix into each PCR vessel. For the samples of interest, add 5 µL sample DNA. For the negative control, add 5 µL H 2 O, PCR-

grade (vial 5, colorless cap). For the positive control, add

5 µL foodproof E. coli O157 control template.

(2) Seal the PCR vessels accurately with optical caps

or foil. Briefly centrifuge the PCR vessels in a suitable centrifuge. Cycle the samples as described below:

Preincubation.1 cycle. Step 1.37°C for 4 min. Step 2.95°C for 5 min. Amplification.50 cycles. Step 1.95°C for 5 s. Step 2 (fluorescence detection in Step 2).60°C for 60 s.

Interpretation and Test Result Report

Description of interpretation procedure and how results are reported.—The amplification of DNA of E. coli O157 was analyzed in the fluorescence channel suitable for FAM-labeled probes detection. The specific amplification of the internal control was analyzed in the fluorescence channel suitable for VIC/HEX. The results

Table 7.

Distribution of inclusivity strains

Serovar

No. strains

E.

coli O157:H7

38

E.

coli O157:H-

19

E.

coli O157:H16

2

E.

coli O157, H unknown

1

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Junge et al.: Journal of aoaC InternatIonal Vol. 94, no. 6, 2011

Table 8.

Inclusivity strain number and origin

Table 8.

(continued)

 

Strain No. (internal)

Serotype

Origin/source

Strain No. (internal)

Serotype

Origin/source

4735

O157:H-

Unknown Human feces Human Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Milk Sausages Minced meat (cattle) Bovine feces Bovine feces Hamburger Raw milk Human feces Ground beef Milk

14248

O157:H7

Human feces

4738

O157:H7

14249

O157:H7

Human feces

4946

O157:H7

14250

O157:H7

Human feces

4948

O157

14251

O157:H7

Human feces

5579

O157:H7

14252

O157:H7

Human feces

5580

O157:H7

14253

O157:H7

Human feces

5854

O157:H7

14254

O157:H7

Human feces

5855

O157:H-

14255

O157:H7

Salami

7840

O157:H-

14256

O157:H7

Ground beef

7842

O157:H-

14257

O157:H7

Ground beef

7844

O157:H-

14258

(NCTC 12079)

O157:H7

Human feces

7848

O157:H-

14259

O157:H-

Human feces

7851

O157:H-

14260

O157:H-

Sausages

7852

O157:H-

14261

O157:H-

Ground beef

7854

O157:H7

14262

O157:H16

Ground beef

7855

O157:H7

14263

O157:H-

Raw milk

7875

O157:H-

14264

O157:H-

Intestine, sheep

8275

O157:H7

14265

(NCTC 12080)

O157:H-

Human feces

8325

O157:H7

 

12503

O157:H-

12507

O157:H-

12518

O157:H7

were compared from channel FAM (E. coli O157) and channel VIC/HEX (internal control) for each sample, and the results were interpreted as described in Table 3.

12538

O157:H7

14173

O157:H7

14174 (ATCC 43895)

O157:H7

Summary of Results

 

14175

O157:H7

 

14176

O157:H-

Internal Comparison Study

 

14177

O157:H16

The alternative PCR method was performed on two different real-time PCR instruments—LightCycler 480 and Mx3005P—with the same results (Table 4). The foodproof E. coli O157 detection kit in combination with the foodproof ShortPrep II kit successfully detected low and high numbers of E. coli O157 in food samples. All uninoculated food samples were negative; no false-positive results occurred. The results of the three low and high inoculated food samples showed a very high correlation with the PCR method and the cultural FDA-BAM or USDA/FSIS methods. Calculation of performance indicators.The four performance indicators for qualitative methods are sensitivity, specificity, false-positive rate, and false- negative rate. The definitions of performance indicators are as follows:

14178

O157:H-

14190

O157:H7

Human feces

14200

O157:H7

Minced meat (cattle)

14211

O157:H7

Minced meat (cattle)

14226

O157:H7

Bovine feces

14227

O157:H7

Bovine feces

14240

O157:H7

Bovine feces

14241

O157:H7

Bovine feces

14242

O157:H7

Human feces

14243

O157:H7

Human feces

14244

O157:H7

Human feces

14245

O157:H7

Human feces

14246

O157:H7

Human feces

14247

O157:H7

Human feces

Sensitivity.—Sensitivity or inclusivity is the ability of the method to detect E. coli O157 from a wide range of

Junge et al.: Journal of aoaC InternatIonal Vol. 94, no. 6, 2011

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Table 9.

Exclusivity strains

No.

Organism

Strain No. (internal)

Strain No. (external)

Strain origin/source

1

Citrobacter koseri

4958

DSM 4595

Throat Child’s throat Spinal fluid Meat (lamb) Minced meat (cattle) Beef Raw milk Minced meat (cattle) Minced meat (cattle) Raw milk cheese Unknown Unknown Unknown Minced meat (cattle) Unknown Unknown Meat (lamb) Unknown Unknown

2

Cronobacter sakazakii

4955

DSM 4485

3

Enterobacter cloacae subsp. cloacae

15136

DSM 30054

4

E. coli O7:H- (STEC)

12509

LM 1126

5

E. coli O8:H- (STEC)

12512

LM 1364

6

E. coli O8:H27 (STEC)

12504

LM 841

7

E. coli O17:H- (STEC)

12505

LM 872

8

E. coli O22:H- (STEC)

12506

LM 1046

9

E. coli O22:H8 (STEC)

14206

1608

10

E. coli O23:H15 (STEC)

12511

LM 1328 H 2459/96/1 H 73/96/1 H 2955/96/1 LM 1394 H 509/95 H 774/89 LM 1119 EH VUB 60

11

E. coli O26:H- (STEC)

5856

12

E. coli O26:H11 (STEC)

5853

13

E. coli O26:H11 (STEC)

5858

14

E. coli O46:H- (STEC)

12514

15

E. coli O48:H21 (STEC)

5852

16

E. coli O55:H- (STEC)

5851

17

E. coli O84:H21 (STEC)

12508

18

E. coli O101:H9 (STEC)

5647

19

E. coli O103:H2 (STEC)

5648

7828/95

20

E. coli O103:H2 (STEC)

5857

H 2947/96/1

Unknown Minced meat (cattle) Unknown Milk Meat (lamb) Minced meat (cattle) Human wound Human clinical isolate Water Human stool Unknown

21

E. coli O104:H12 (STEC)

12515

LM 1398

22

E. coli O111:H2 (STEC)

5849

H 946/87/1

23

E. coli O138:H8 (STEC)

12502

LM 680

24

E. coli Ont:H- (STEC)

12510

LM 1247

25

E. coli Orauh:H23 (STEC)

12513

LM 1389

26

Escherichia vulneris

5611

DSM 4564

27

Hafnia alvei

8930

DSM 30163

28

Klebsiella pneumoniae subsp. pneumoniae

4957

DSM 30102

29

Salmonella enterica subsp. enterica (Enteritidis)

14151

2627/00

30

Shigella flexneri

2144

DSM 4782

strains in an as-small-as-possible amount of CFU/25 g food sample. Sensitivity rate (p+) for a food type and inoculation level is defined as the probability that a

method, alternative or reference, will classify a test sample as positive, given that a test sample is a known positive.

A known positive refers to the confirmation of inoculated

analyte. Sensitivity rate is defined as the total number of confirmed positive test portions by the method divided by total number of confirmed positive test portions by both the alternative and reference methods. Specificity.—Specificity or exclusivity is the lack of interference in the alternative method from a relevant range of nontarget strains, which are potentially cross-reactive. Specificity rate (p+) for a food type and inoculation level

is defined as the probability that a method, alternative or

reference, will classify a test sample as negative, given

that a test sample is a known negative. A known negative refers to a confirmed negative test portion. Specificity rate is defined as the total number of confirmed negative test portions by the method divided by total number of confirmed negative test portions by both the alternative and reference methods. Repeatability.—The repeatability is within-laboratory precision, designated s r , or the closeness of agreement between successive and independent results obtained by the same method on identical test material, under the same conditions (e.g., apparatus, operator, laboratory, and incubation time). Stability.—Stability is defined as consistent detection results of Salmonella without influence of different production lots or shelf life. In the following the combined results of the whole

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Junge et al.: Journal of aoaC InternatIonal Vol. 94, no. 6, 2011

repeatability study (in-house and independent validation studies) for these four performance indicators are provided:

Low inoculation level (1–10 CFU/25 g).—Sensitivity rate (p+) = 18/19 = 0.95; specificity rate (p–) = 42/41 = 1.02; false-positive rate (pf+) = 0/41 = 0; false-negative rate (pf–) = 1/20 = 0.05. High inoculation level (10–50 CFU/25 g).— Sensitivity rate (p+) = 40/39 = 1.03; specificity rate (p–) = 20/21 = 0.95; false-positive rate (pf+) = 1/22 = 0.045; false-negative rate (pf–) = 0/39 = 0. Test of significance difference (χ 2 ).—The alternative method must be statistically equivalent to the reference method for each food matrix and each inoculation level. Equivalence is measured using the Chi-square (χ 2 ) methodology. For qualitative methods the McNemar test is conducted. A Chi-square value <3.84 indicates that the hypothesis that the test method and reference method are equivalent could not be rejected at the 5% level of confidence. This criterion must be satisfied for each level of each food type. Chi-square, as defined by McNemar, is:

of each food type. Chi-square, as defined by McNemar, is: where a = test samples positive

where a = test samples positive by the alternative method and negative by reference method, b = test samples negative by the alternative method and positive by reference method. Tables 5 and 6 show the Chi-square results for each food type, each table for one inoculation level. The criterion of a Chi-square value <3.84 was achieved for each level of each food type.

Specificity

Inclusivity.—The objective was to verify on both real-time PCR instruments, the LightCycler 480 and the Stratagene Mx3005P, that different strains of E. coli of serotype O157 are detected with the foodproof E. coli O157 Detection Kit, 5′ Nuclease. Sixty strains with known serotype O157 were tested with the foodproof E. coli O157 Detection Kit. The tested strains belonged to the serovars listed in Tables 7 and 8. The foodproof E. coli O157 Detection Kit detected various isolates of E. coli of serotype O157 with both real-time PCR instruments. No false-negative results occurred. Exclusivity.—The objective was to verify on both

real-time PCR instruments, the LightCycler 480 and the Stratagene Mx3005P, that organisms other than E. coli O157 are not detected with the foodproof E. coli O157 Detection Kit, 5′ Nuclease. Thirty strains of nontarget organisms were tested, including E. coli of other serotypes as well as strains of the same microbiological environment. The suitability of the extracts for PCR are shown with a consensus-PCR system (Table 9). The foodproof E. coli O157 Detection Kit was specific for E. coli of serotype O157 on both real-time PCR instruments. No false-positive results occurred.

Conclusions

For this method extension a repeatability study/method comparison with three different food matrixes was accomplished. Moreover the inclusivity and exclusivity of the real-time PCR system were examined with a wide spectrum of different isolates. Therefore the BIOTECON Diagnostics foodproof E. coli O157 Detection Kit in combination with the foodproof ShortPrep II kit was tested on two different real-time PCR instruments, the LightCycler 480 System from Roche Diagnostics and the Mx3005P from Agilent/Stratagene. The repeatability study and the inclusivity and exclusivity studies gave the expected results. No deviations occurred, and all results were within the expected range.

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Centers for Disease Control and Prevention, http://www.

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cdc.gov/ncidod/dbmd/diseaseinfo/escherichiacoli_g.htm U.S. Food and Drug Administration (2011) Bad Bug

BookEscherichia coli O157, http://vm.cfsan.fda.

(3)

Karmali, M.A. (1989) Clin. Microbiol. Rev. 2, 15–38.

doi:10.1006/fmic.1997.0134

(4)

Scheu, P.M., Berghof, K., & Stahl, U. (1998) Food

http://www.cfsan.fda.gov/~ebam/bam-4a.html

(5)

Microbiol. 15, 13–31 Grant, M.A. (2004) Appl. Environ. Microbiol. 70,

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