Version 1: 7/2/02

1

AOAC Validation Report

RapidChek® for Rapid Detection of Escherichia coli O157 (including H7) in Raw Boneless

Beef, Raw Ground Beef and Apple Cider

Version 1: 7/2/02

2

RapidChek® for Rapid Detection of Escherichia coli O157 (including H7) in Raw Boneless Beef, Raw Ground Beef and Apple Cider

Abstract Two different food types, raw beef and apple cider, which are generally regarded as significant vectors of Escherichia coli O157 were analyzed using the novel lateral flow device (LFD), RapidChek, for the detection of this organism. The LFD Test was utilized in conjunction with an 8 h and 18 h enrichment protocol using the proprietary resuscitation media, RapidChek 8-Hour E. coli Media, for maximal recovery of E. coli O157 in these specified foods. The LFD Test was also combined with traditional enrichment protocols (mEC and EEB) for a 20 h test and all methods were compared with either the USDA/FSIS or the FDA/BAM (reference) culture methods for the detection of E. coli O157:H7. Raw boneless beef, raw ground beef, and apple cider were inoculated with E. coli O157:H7 at two levels, a high level, where predominantly positive results were anticipated, and a low level, where fractional recovery was expected. In these studies, 400 samples artificially inoculated with E. coli O157:H7 and 50 non-inoculated controls were analyzed using the LFD test method. The LFD Test reported 373 presumptive positives, of which, 373 were confirmed as positive by either the USDA/FSIS or FDA/BAM. All non-inoculated controls tested negative using the LFD Test method. In comparison, the reference methods detected 97 of the 120 artificially contaminated samples as positive for the organism. As a result the overall agreement between the LFD Test and reference methods averaged 87%. The agreement between methods was significantly influenced by the poor recovery of the reference method in one of the matrices. No false positive results were reported by the LFD Test Method. The use of the RapidChek test with RapidChek Media or mEC and EEB enrichment media has been shown to perform as well as the USDA/FSIS and FDA/BAM methods for the detection of E. coli O157 in raw ground and boneless beef, and apple cider. Method Authors Orla Cloak, Tim Lawruk, Jingkun Li, and George Teaney Strategic Diagnostics Inc., 128 Sandy Drive, Newark, Delaware 19713-1147 Submitting Company Strategic Diagnostics Inc., 111 Pencader Dr., Drive, Newark, Delaware 19702 Independent Laboratories Silliker Corporate Research Center, 160 Amory Drive, South Holland, IL 60473 Reviewers 1.0 Scope of Method

1.1 Target organisms – Escherichia coli O157 (including H7)

Version 1: 7/2/02

3

1.2 Matrices – Raw boneless beef, raw ground beef and apple cider

1.3 Summary of validated performance claims – The LFD Test Method for E. coli O157 was

evaluated and was shown to have 100% specificity (exclusivity) and 100% sensitivity (inclusivity) in this study. The accuracy of the LFD Test used with the Proprietary enrichment media for raw ground beef, raw boneless beef, and apple cider averaged 100%. Similarly, the accuracy of the LFD Test used with mEC and EEB media for the detection of E. coli O157 in raw ground and boneless beef, and apple cider averaged 100%. The method agreement between the LFD Test Methods and the USDA/FSIS and FDA/BAM methods for the detection of E. coli in raw ground and boneless beef and apple cider averaged 87%.

2.0 Definitions

2.1 Sensitivity – Rate of sensitivity = (No. of LFD Test Positives)/(No. Tested) × 100.

2.2 Specificity – Rate of specificity = (No. of LFD Test Negative)/(No. Tested) × 100.

2.3 Accuracy – (No. of Confirmed Positives/Total No. Positives Tested) × 100.

2.4 Method Agreement. – Method Agreement = [1- (No. of Correct LFD Test – No. of Correct Reference Method / Total No. Method Samples)] × 100.

3.0 Principle

3.1 The LFD Test for E. coli O157 can be used in combination with the proprietary enrichment broth for a rapid 8 hour test or an 18 hour test. After suitable enrichment, the sample broth is dispensed into the sample port of the LFD. The sample flows through a zone containing antibody coated colloidal gold reagents specific to E. coli O157. If antigens are present in the sample, they will bind to the antibody-conjugates to form an antigen/antibody complex. As this complex migrates through the nitrocellulose matrix, it passes a zone of anti-E. coli O157 antibody. If antigen is present, the complex is captured in this zone and is visualized by the formation of a red line. A second zone on the membrane is designed to capture any antibody-gold complex not bound in the first zone. As a result, when E coli O157 antigen is present, the formation of 2 red lines is observed, whereas when no E. coli O157 is present, only 1 line forms.

4.0 General Information

4.1 Escherichia coli O157 was first isolated in 1976 (1), but was not identified as a human pathogen until 1982, after an outbreak of hemorrhagic colitis associated with consumption of undercooked beef patties (2). Since this time, it has been recognized as a significant cause of foodborne infection (3). Symptoms of E. coli O157 infections can vary from mild non bloody diarrhea to bloody diarrhea with severe abdominal pain (hemorrhagic colitis)(4). Susceptible individuals, including those at extremes of life and the immunocompromised, can develop more serious complications including hemolytic uremic syndrome (HUS), the leading cause of renal failure in

Version 1: 7/2/02

4

children (5) and this occurs in about 5% of cases and thrombocytopaenic purpura, a neurological disorder. Early recognition of E. coli O157 infections is therefore critical for effective treatment of patients. Cattle have been identified as a major reservoir of the organism and foods of bovine origin, including beef and dairy products have been implicated in many outbreaks of infection worldwide (6,7). More recently, apple cider, a traditional unpasteurized, acidic beverage, has been linked to E. coli O157 infections (8, 9). Although, E. coli O157:H7 cannot grow in apple cider, it has a particularly high acid tolerance and can survive for prolonged periods of time in this food matrix (8). Since E. coli O157 has an extremely low infective dose, with fewer than 50 viable cells capable of causing illness (10), it is particularly pertinent that it can be detected in foods when present in low levels. Thus, sensitive, fast and reliable microbial detection methods are needed in the food industry to monitor foods for E. coli O157, and ensure that safe food is being supplied to the consumer.

5.0 Test Kit Information

5.1 Kit Name – RapidChek E. coli O157 test kit, includes 50 lateral flow devices, with the option of a proprietary RapidChek Media for E. coli enrichment media.

5.2 Catalog Number – 7000160

5.3 Ordering Information – Strategic Diagnostics Inc., 111 Pencader Drive, Newark, DE 19702;

Phone: (800) 544-8881 or www.sidx.com

6.0 Media and Reagents

6.1 RapidChek E. coli O157 Lateral Flow assay

6.2 Enrichment –

6.2.1 RapidChek Media for E. coli– (Strategic Diagnostics Incorporated, Newark, DE). Dissolve 25.2g in one liter of sterile distilled or deionized water prewarmed to 42oC. Use immediately.

6.2.2 Modified EC Broth (mEC) - (Difco Becton Dickinson). Dissolve 36.6g in one liter of

sterile distilled or deionized water. Autoclave for 15 minutes at 121oC. Cool to room temperature. Add 20mg/L of novobiocin (Sigma, St. Louis, MO).

6.2.3 Enterohaemorraghic E. coli Broth (EEB) – modified tryptone soya broth (30g tryptone

soya broth, 1.5 g of dipotassium phosphate, 1.5g of bile salts #3) per one liter of distilled or deionized water and autoclaved for 15 minutes at 121°C. The broth was cooled to room temperature and supplemented with novobiocin (20mg/L), cefixime (0.05 mg/L), vancomycin (10mg/L) and cefsulodin (8mg/L).

Version 1: 7/2/02

5

6.3 Diagnostic reagents - Culture and biochemical reagents necessary for traditional confirmation of presumptive positives. See “Chapter 5. Detection, Isolation and Identification of Escherichia coli O157:H7 and O157:NM (Nonmotile) From Meat and Poultry Products” in USDA/FSIS Microbiology Laboratory Guidebook , 3rd Edition, Rev #1, 9/6/99. Web page: http://www.fsis.usda.gov/ophs/microlab/mlgbook.html

6.4 Disposable 200 µl pipette tips

6.5 Filtered stomacher bags

6.6 MACS Large Cell Separation Columns (Miltenyi Biotec, Auburn, CA) 6.7 OctoMACS Separation Magnet and Stand (Miltenyi Biotec) 6.8 Sterile 50ml conical tubes (Falcon) 6.9 40 µm Cell Strainer (Falcon)

7.0 Apparatus

7.1 RapidChek E. coli O157 Lateral Flow devices. See figure 1 7.2 Hot plate for heat - killing enriched samples. 7.3 Microcentrifuge for concentrating cells. 7.4 Micropipet – Capable of accurately dispensing 150 µl. 7.5 Shaking incubator – Capable of holding temperature at 42oC and shaking at 120 rpm for 8hours. 7.6 Stationary incubator – Capable of holding temperature at 37oC for 20-24 hours. 7.7 Stomacher – Seward 400 Circulator (Seward, London, England) - for thorough mixing of food

samples in enrichment broth. 7.8 Top loading balance – To weigh 25 g ( + 0.1g) test samples.

8.0 Standard Reference Material

None

Version 1: 7/2/02

6

9.0 Safety Precautions

9.1 Live bacteria may be infectious and pose a significant health hazard, E. coli O157, in particular, has a low infective dose, approximately 50 cells capable of causing illness. All bacterial cultures should be handled with caution and biohazard waste should be disposed of appropriately.

10.0 Sample Preparation

10.1 Raw boneless beef, raw ground beef and apple cider

10.1.1 Aseptically weigh a representative 25 g or 25 mL sample and add to filtered stomacher bag.

10.1.2 Add 225 ml of mEC (for beef), EEB (for cider) pre-warmed to 35 oC or RapidChek

Media pre-warmed to 42 oC.

10.1.3 Stomach samples for approximately 45 seconds.

10.1.4 Incubate mEC or EEB samples at 37oC for 20-24 h in a stationary incubator for the USDA reference method. Incubate Proprietary Media samples at 42oC for 8 h without shaking (boneless beef only), 8 h with shaking at 120 rpm, or 18 h (not shaken) for testing with the LFD Test.

10.2 Retain the remainder of the enriched sample for culture confirmation of presumptive positive

results.

11.0 Set Up and Operation

11.1 Remove the required number of test cassettes from the foil pouch and place on a flat surface. 11.2 Label each cassette with the appropriate sample identification. 11.3 Take one pipette from the bag. Squeeze and hold the bubble on the top of the pipette and place

in the sample enrichment (samples enriched either in the Proprietary Media (8 h or 18 h), mEC (20 h) or EEB broth (20 h).

11.4 Release the bulb and the required volume of solution (150uL) will rise in the barrel of the

pipette. 11.5 Hold the pipette over the sample reservoir on the cassette and add the entire sample volume of

the pipette barrel by squeezing the bulb. Discard the used pipette. 11.6 Let the cassette develop for 10 minutes.

Version 1: 7/2/02

7

12.0 Interpretation and Test Result Report

12.1 After a 10 minute time period, the appearance of one red line (control line) on the LFD Test indicates a negative result. The appearance of two red lines ( the control line and the test line) on the LFD Test indicates a positive result (See Figure 1).

13.0 Independent Validation Studies

13.1 Validation studies were conducted by Silliker Laboratories in South Holland, Illnois, under the direction of the AOAC Research Institute.

13.2 Method Comparison Study - Raw ground beef was analyzed by the LFD Test to determine the

agreement of the kit performance relative to the current reference method for this food matrix. Replicate samples at three inoculum levels (zero, low and high) were examined for each matrix. Refer to Table 4 for a summary of the independent validation study results.

13.2.1 Methodology

13.2.1.1 Raw ground beef

13.2.1.1.1 E. coli O157 (ATCC # 35150) was grown in 10 ml TSB broth for

at 37°C for 18 h. Twenty five hundred grams of raw ground beef was inoculated with a low inoculum (low = 1-10 cfu/25g) or a high inoculum (high = 10-50 cfu/25g) and homogenized by shaking in a large stomacher bag for 5 minutes. Samples were stressed, to mimic real life environmental conditions, by storing the batch of ground beef at 4oC for 48 h. An MPN (most probable number) was set up to determine cell levels after the stress period. Meat from each inoculum level was divided into four sets of twenty 25 g samples. One set from each inoculum level was stomached with 225 ml of either the Proprietary Test Media or mEC for 45 seconds. Two sets of each inoculum was enriched in the Proprietary Test Media for either 8 h with agitation or for 18 h without agitation. Two additional sets of each inoculum level were enriched in mEC for 20 h, with one set of these samples being examined on the LFD Test and the second set was processed according to the USDA/FSIS reference protocol for E. coli O157 (10).

13.2.1.1.2 All positive analysis were confirmed using the standard cultural

and biochemical tests recommended by FSIS/USDA. In brief, this involves immunomagnetic separation with dynal beads specific for E. coli O157 from each sample with plating onto Rainbow agar. Typical E. coli O157 colonies were subsequently tested with latex agglutination test and latex positive colonies were streaked to 5%

Version 1: 7/2/02

8

sheeps blood agar plates. These cultures were further confirmed by testing for motility, cellobiose, TSI and API20E.

13.2.1.1.3 One set of the mEC (reference set) were screened by an ELISA

(Neogen) and subsequently confirmed by the FSIS/USDA confirmation tests described previously (10).

13.3.1 Results

13.3.1.1 The results from the external laboratory study reported excellent accuracy and method agreement for the LFD Test -8 Proprietary Test Media, LFD Test-18 hour Proprietary Test Media and The LFD Test-20 hour mEC methods (Table 4). All non-inoculated samples were determined to be negative in all methods. The LFD Test-8 Proprietary Test Media method recovered 39 of the 40 inoculated samples and confirmed all 39 of the recovered samples (100% overall accuracy) with 100% method agreement. LFD Test-18 Proprietary Test Media method recovered and positively identified 40 of the 40 inoculated samples resulting in 100% accuracy with 97% agreement with the reference method. Lastly, the LFD Test-20 hour mEC method recovered 39 of the 40 inoculated samples and confirmed all 39 positives resulting in an overall method accuracy of 100% and an overall method agreement of 100%.

14.0 Internal Validation Studies

14.1 Inclusivity Study

14.1.2 Fifty E. coli O157 strains (Table 1) were analyzed to determine the sensitivity and specificity of the LFD Test.

14.1.3 Methodology

14.1.3.1 Fifty E. coli O157 strains were inoculated into individual 10 ml aliquots of the

Proprietary Test Media and incubated at 42oC for 18 h. After enrichment, samples were tested on the LFD Test as described previously.

14.1.4 Results

14.1.4.1 Results from the Inclusivity study are summarized in Table 3. As can be seen, the LFD Test was found to have a sensitivity of 100% for detecting specific strains of E. coli O157 from a variety of sources including clinical, environmental and food isolates.

14.2 Exclusivity Study

14.2.2 Thirty one non-E. coli O157 strains (Table 3) were tested to determine the specificity of

the kit.

Version 1: 7/2/02

9

14.2.3 Methodology

14.2.3.1 Strains were grown at 37 oC for 18 h in 10 ml of TSB broth. After incubation, samples were evaluated using the LFD Test as described previously.

14.2.4 Results

14.2.4.1 Exclusivity results are summarized in Table 3. Of the thirty one non-E. coli

O157 strains tested, all thirty one were correctly identified as negative by the LFD Test, resulting in a specificity of 100%.

14.3 Method Comparison Study

14.3.2 Raw boneless beef and apple cider were analyzed by the LFD Test to determine the

agreement of the kit performance relative to the current reference method for each food matrix examined. Replicate samples at three inoculum levels (zero, low and high) were examined for each matrix.

14.3.3 Methodology

14.3.3.1 Raw boneless beef

14.3.3.1.1 E. coli O157 (ATCC # 35150) was grown in 10 ml TSB broth for

at 37°C for 18 h. Twenty five hundred grams of boneless beef was inoculated with a low inoculum (low = 1-10 cfu/25g) or a high inoculum (high = 10-50 cfu/25g) and homogenized by shaking in a large stomacher bag for 5 minutes. Samples were stressed, to mimic real life environmental conditions, by storing the batch of boneless beef at 4oC for 48 h. An MPN (most probable number) was set up to determine cell levels after the stress period. Pre-cold stress and post-cold stressed inoculum plate counts were conducted to verify inoculum levels. Meat from each inoculum level was divided into four sets of twenty 25 g samples. One set from each inoculum level was stomached with 225 ml of either the Proprietary Test Media (pre-warmed to 42 oC) or mEC (pre-warmed to 35 oC)for 45 seconds. Three sets of each inoculum were enriched in the Proprietary Test Media for either 8 h with and without agitation, or for 18 h without agitation. Two additional sets of each inoculum level were enriched in mEC for 20 h, with one set of these samples being examined on the LFD Test and the second set was processed according to the USDA/FSIS reference protocol for E. coli O157 (10).

14.3.3.1.2 All positive analysis were confirmed using the standard cultural

and biochemical tests recommended by FSIS/USDA. In brief, this

Version 1: 7/2/02

10

involves immunomagnetic separation with dynal beads specific for E. coli O157 from each sample with plating onto Rainbow agar. Typical E. coli O157 colonies were subsequently tested with latex agglutination test and latex positive colonies were streaked to 5% sheeps blood agar plates. These cultures were further confirmed by testing for motility, cellobiose, TSI and API20E.

14.3.3.1.3 One set of the mEC (reference set) were screened by an ELISA

(Neogen) and subsequently confirmed by the FSIS/USDA confirmation tests described previously (10).

14.3.3.2 Apple Cider

14.3.3.2.1 E. coli O157 (ATCC #35150) was grown in 10 ml of TSB broth at

37oC for 18 h. Twenty five hundred milliliters of apple cider was inoculated with E. coli O157 at a low level inoculum (1-10 cfu/25ml) or at a high level inoculum (10-50 cfu/25ml) and homogenized by shaking in a large stomacher bag for 5 minutes. Samples were stressed, to mimic real life environmental conditions, by storing the batch of apple cider at 4oC for 48 h. An MPN was set up to determine cell levels after the stress period. Pre-cold stress and post-cold stressed inoculum plate counts were conducted to verify inoculum levels. Apple cider from each inoculum level was divided into four sets of twenty 25 ml samples. One set from each inoculum level was mixed with 225 ml of either the Proprietary Test Media or EEB broth for 45 seconds. Two sets of each inoculum were enriched in the Proprietary Test Media for either 8 h with agitation or for 18 h without agitation. Two additional sets of each inoculum level were enriched in EEB broth for 20 h, with one set of these samples being examined on the LFD Test and the second set was processed according to the FDA/BAM protocol for E. coli O157 (11). All positive analysis were confirmed utilizing the FDA/BAM confirmation tests, in brief, this involves isolation of the organism onto Sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) plates, these plates were incubated overnight at 37oC. Typical E. coli O157 colonies were confirmed by spot indole test, latex agglutination test, and API 20E (11).

14.3.4 Results

14.3.4.1 Results from the method comparison/repeatability study are summarized in Tables 5 - 6. The performance of the LFD Test with the 8 hour shaken and non-shaken Proprietary Media, 18 hour non-shaken Proprietary Media, and 20 hour mEC enrichments of raw boneless beef was found to be comparable to the USDA/FSIS reference method (Tables 5). All non-inoculated samples were found to be negative by both methods. The LFD Test-8 hour shaken and

Version 1: 7/2/02

11

non-shaken Proprietary Media methods recovered and positively identified all 40 of the 40 inoculated samples, whereas the reference method recovered 21 of the 40 inoculated samples. The LFD Test-8 Proprietary Media method reported 100% accuracy in this study, with an overall method agreement of 58%.

14.3.4.2 The LFD-18 hour Proprietary Media method recovered and identified 39 of

the 40 inoculated samples whereas the reference method identified 21 samples (Table 5). All non-inoculated samples were found to be negative by both methods yielding an overall method agreement of 60%.

14.3.4.3 In the final raw boneless beef method comparison, the LFD Test-20 hour

mEC and the USDA/FSIS methods recovered and identified 20 and 21 of the 40 inoculated samples respectively. All non-inoculated samples were found to be negative by both methods yielding an overall method agreement of 98%.

14.3.4.4 In an effort to verify the inoculation rate for the study, pre-cold stress, post-

cold stress and sample MPN evaluations were conducted. Inoculum samples pre- and post cold stressed were plated in triplicate on LB agar. Inoculation levels for the low and high treatments were reported at 5 and 20 cfu/25g prior to the cold stress process and 2.7 and 10.9 cfu/25g following the 48 hour cold stress process (Table 5). A 3 tube MPN analysis of the inoculated raw boneless beef following the 48 hour cold stress period reported 0.4 and 23.3 cfu/25g for the low and high inoculation treatments respectively.

14.3.4.5 Similarly, in the method comparison with apple cider, the LFD Test-8 hour,

18 hour and EEB methods demonstrated a high degree of accuracy and method agreement (Table 6). All non-inoculated samples were determined to be negative in both methods. The LFD Test-8 hour Proprietary Media method recovered 36 of the 40 inoculated samples whereas the reference method recovered 37 samples resulting in an overall method agreement of 98%.

14.3.4.6 The LFD Test-18 hour Proprietary Media method recovered 40 of the 40

sample whereas the reference method recovered 37 samples. All non-inoculated samples were found to be negative by both methods. The resulting method agreement was 93%.

14.3.4.7 In the comparison of the FDA/BAM method with the LFD Test-EEB method,

the reference method recovered and positively identified 37 of the 40 samples, while the LFD Test-EEB method identified and confirmed 40 of the 40 samples as positive (100% accuracy). All non-inoculated samples were found to be negative by both methods. The overall method agreement for this study was 93%.

Version 1: 7/2/02

12

14.4 Ruggedness Studies

14.4.1 The LFD Test is a very simple device to utilize. It is a compact open cassette which

requires the edition of an aliquot of the enriched sample. Therefore, ruggedness testing for this study involved variation in temperature at which the device was tested, variations in times at which the strip was read, minor variations in sample preparation/enrichment times. Ruggedness testing additionally involved variations in sample volume added to the LFD Test, variability in time lots of the test strip used, and stability of test line after expiration of reading time has occurred. A comparison of live versus boiled enrichment samples was also examined to give the customer the option of boiling the aliquot of enriched specimen, if so desired.

14.4.2 Methodology

14.4.2.1 Temperature Latitude Study – LFD Tests were removed from their foil

pouches and placed at 12°C, 20°C and 30°C to mimic potential temperature range at which the devices could be tested by the user. When devices had equilibrated to these temperatures, 5 samples negative for E. coli O157:H7 (non-inoculated samples boneless beef samples, enriched with the Proprietary Media for 18 h) and 5 samples positive for E. coli O157:H7 (three samples inoculated with high levels of E. coli {20 cfu/25g} and two samples inoculated with low levels of the organism {5 cfu/25g}) were tested on the devices at 12°C, 20°C and 30°C. Inoculated boneless beef samples were enriched in the Proprietary Media for 18 h and tested on the devices stored at various temperatures stated above.

14.4.2.2 Reading Device Time Study - It is recommended in the package insert

information for the LFD Test that 10 minutes is required prior to reading the strip and recording the result as negative for E. coli O157. This study was designed to determine what effect a variation on reading the strip had on the end result of the assay. Five samples negative for E. coli O157:H7 (non-inoculated samples boneless beef samples, Proprietary Media enriched for 18 h) and 5 samples positive for E. coli O157:H7 (three samples inoculated with high levels of E. coli {20 cfu/25g} and two samples inoculated with low levels of the organism {5 cfu/25g}) were tested on the devices. LFD Tests were read at 2 minute intervals over a period of 20 minutes.

14.4.2.3 Sample Enrichment Time Study – Inoculum, for the Proprietary Media

enriched samples (8 h and 18 h), was grown in 10 ml of TSB as described previously (E. coli O157 (ATCC # 35150)). Two sets of samples were set up. consisting of five 25 g samples of boneless beef samples inoculated with 5 cfu/25g, five 25g samples of boneless beef inoculated with 20 cfu/25g and two 25 g samples were non-inoculated. Samples were enriched in 225 ml of the Proprietary Media and aliquots of 150 uL were removed at 7 h, 8 h and 9 h, and tested using the LFD Test. A second set of samples were set up as

Version 1: 7/2/02

13

described in the previous sentences, and enriched in 225 ml of the Proprietary Media for 17 h, 18 h and 19 h. Aliquots of 150ul were removed from the enrichment cultures at these time intervals and tested using the LFD Test.

14.4.2.4 Sample Volume Study - It is recommended in the package insert for the LFD

Test, that 150µl of the enriched sample be added to the port of the device. This study determined the effect of variations in the sample volume added to the device. One positive (1-10 cfu/25g) and one negative (0 cfu/25g) Proprietary Media enriched boneless beef sample was tested on the device. Volumes of 100µl, 200µl, 300µl, 400µl and 500µl of negative and positive samples were added to the device. LFD Tests were run in duplicate and cassettes were opened after 10 minutes had expired and observations were made.

14.4.2.5 Lot to Lot Variability Study - Boneless beef samples were inoculated with E.

coli O157:H7 at two levels (high and low, 20 samples at each level) and enriched in the Proprietary Media for 18 h at 42°C. Five non-inoculated controls were run with this experiment. After incubation, samples were tested on three production lots of the LFD Tests to determine the lot to lot variability of the strips.

14.4.2.6 Stability Study - The stability of the LFD Test was examined over a period of

46 days. LFD Tests were stored at 4°C, 25°C, 37°C and 45°C under desiccated conditions. Devices were removed from their respective storage temperatures and tested with boneless beef samples inoculated with a low level of E. coli O157 and a high level of E. coli O157. An non-inoculated boneless beef control was also examined. Samples were tested on the LFD Test at the following time points, day 0, 7, 14, 21, 28, 35, 46.

14.4.2.7 Boiling Versus Live Sample Study - Users of the system may prefer to boil

samples prior to the addition to the LFD Test. Therefore, a study was carried out to compare boiled aliquots of the enriched sample and live aliquots of the enriched sample. Ground beef samples were set up, including 10 samples inoculated with a low level of E. coli O157, 10 samples inoculated with a high level of the organism, and five non-inoculated controls. One set of samples was enriched in the Proprietary Media for 8 h, and a second set was enriched in mEC broth for 20 h. After enrichment samples were tested live on the LFD Test and a 10 ml aliquot of each sample was removed, placed in a boiling water bath and boiled for 15 minutes. Samples were removed from the boiling water bath, allowed to cool to room temperature and subsequently tested on the device.

14.4.3 Results

14.4.3.1 Results from the ruggedness studies are summarized in Tables 7-12. The LFD

Test was found to function properly throughout the temperature range

Version 1: 7/2/02

14

reported (Table 7). Additionally, when interpreting the test result in the reported temperature range, the test result should be evaluated between 10 and 20 minutes following the addition of sample (Table 7). Devices showing positive results as soon as 4 minutes can be reported as positive. In order to properly identify negative samples, the manufacture recommends reading the device as soon as 10 minutes following the addition of sample.

14.4.3.2 The latitude around the timing of the sample enrichment was evaluated for the

Proprietary Media 8 hour and 18 hour enrichments. The duration of Proprietary Media enrichments was shown to have at least +/- 1 hour latitude in the 8 hour and 18 hour methods (Table 8).

14.4.3.3 The volume of sample applied to the lateral flow device did not appear to

adversely impact the end result of the test (Table 9). However, sample volumes greater than 250uL will impact the clarity and consistency of the assay line development. Therefore the manufacture recommends the use of 150uL disposable pipette supplied in the LFD kit.

14.4.3.4 The variability of the LFD Test was evaluated across 3 production lots of the

LFD product (Table10). In this evaluation, no significant lot to lot variability was observed with the LFD product.

14.4.3.5 The stability of the LFD Test was evaluated in a short term accelerated

stability study sampling periodically over a 46 day period (Table 11, select data reported). The LFD Test demonstrated exceptional stability at 45oC for at least 46 days. These results strongly suggest that this product will have 1 year stability at room temperature an possibly longer duration under refrigerated conditions.

14.4.3.6 Lastly, boiling of the sample was evaluated as an alternate sample preparation

method (Table 12). Results from this study report 98% agreement between live and boiled samples. The 1 discrepant result was found in an inoculate, mEC enriched sample which was found to be negative as a live sample and positive as a boiled sample.

14.0 Discussion

14.1 The use of the LFD Test with the Proprietary enrichment media has demonstrated excellent accuracy , sensitivity and specificity throughout this study. The overall accuracy of these methods was 100% with 100% sensitivity and specificity. Though excellent method agreement was reported for raw ground beef testing and apple cider testing, the method agreement was slightly lower for the raw boneless beef enriched with the Proprietary Media and tested with the LFD Test. It is believed that this is a direct reflection of the enhanced resuscitation and expansion qualities, and reproducible performance of the Proprietary Media.

Version 1: 7/2/02

15

14.2 The moderate recovery of the USDA/FSIA reference method with the boneless beef has been

observed in this laboratory as well as other laboratories conducting similar studies16.12 . This appears to be primarily a function of the resuscitation qualities of the mEC media and not a function of reference method ELISA and subsequent confirmation methods. Though speculative, one working theory is that the aerobic microbial background population in the beef sample will influence the mEC media resuscitation and recovery of E. coli O157 from cold stressed conditions. Total aerobic microbial counts for the boneless beef in this study exceeded 4E4 cfu/g. The total microbial background reported by the external laboratory for the raw ground beef was 110 cfu/g. In a limited sampling of studies from our laboratory, we’ve seen an inverse correlation between the number of spiked samples recovered from an mEC enrichment and the level of aerobic microbial background.

15.0 Conclusions

15.1 The LFD Test, when used with the Proprietary Media in 8 and 18 hour enrichment methods, has demonstrated excellent sensitivity and selectivity (100%), and 100% accuracy in raw ground and boneless beef and apple cider. The LFD Test has also demonstrated equivalent performance in mEC and EEB media relative to the respective reference methods.

16.0 Reference

16.1 Riley, L.W., Remis, R.S., Helgerson, S. D. et al (1983) Hemorrhagic colitis associated with a

rare Escherichia coli serotype. New England Journal of Medicine, 308, 681-685. 16.2 Parry, S.M. and Palmer, S.R. (2000) The public health significance of VTEC O157. Journal of

Applied Microbiology Symposium Supplement, 88, 1S-9S.

16.3 Dundas, S. and Todd, W.T.A. (2000) Clinical presentation, complications and treatment of infection with verocytotoxin-producine Escherichia coli. Challenges for the clinician. Journal of Applied Microbiology Symposium Supplement, 88, 24S-30S.

16.4 Bell, B.P., Griffin, P.M., Lozano, P., Christie, D.L., Kobayashi, J.M. and Tarr, P.I. (1997)

Predictors of hemolytic uremic syndrome in children during a large outbreak of Escherichia coli O157:H7 infections. Pediatrics 100, 12.

16.5 CDC (1993) From the Centers of Disease Control and Prevention: A multistate outbreak of

Escherichia coli O157:H7 infections from hamburgers – Western United States, 1992 – 1993. The Journal of American Medical Association 269, 2194-2196.

16.6 Upton, P. and Coia, J. E. (1994) Outbreak of Escherichia coli O157 infection associated with

pasteurised milk supply. Lancet 344, 1015.

Version 1: 7/2/02

16

16.7 Garland-Miller, L., and Kasper, C. (1994) Escherichia coli O157:H7 acid tolerance and survival in apple cider. Journal of Food Protection 57, 460-464.

16.8 Besser, R.E.S., Lett, S.M., Weber, J.T., Doyle, M.P., Barrett, T.J., Wells, J.G. and P.M., Griffin.

(1993) An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia coli O157:H7 in fresh pressed apple cider. JAMA 269, 2217-2220.

16.9 Buchanan, R., and Doyle, M. (1997) Foodborne disease significance of Escherichia coli

O157:H7 and other enterohemorrhagic E. coli. Food Technology 51, 69-76.

16.10 Cray, W.C., Abbot, D.O., Beacorn, F.J. and Benson, S.T. (1998) Chapter 5; revision 2: Detection, isolation and identificaiton of Escherichia coli O157:H7 and E. coli O157:NM from meat products. USDA/FSIS Microbiology Laboratory Guidebook , 3rd Edition, 1998, Volumes 1 & 2, U.S. Government Printing Office, Washington, D.C. http://www.fsis.usda.gov/ophs/microlab/mlgbook.html

16.11 Hitchins, A.D., Feng, P., Watkins, W.D., Rippey, S.R. and Chandler, C.A. (1998) Chapter 4 :

Escherichia coli and coliform bacteria. Food Drug and Admistraion/Bacteriological Analytical Manual 8th edition, revision A. http://www.cfsan.fda.gov/comm/microbio.html

16.12 Bird, Charls B., and Rebecca J. Hoerner. (2001) Comparison of the Reveal 20-hour method and

BAM culture method for the detection of Escherichia coli O157:H7 in select foods and environmental swabs: collaborative study. Journal of AOAC International Vol. 84, No. 3, 737-751.

Version 1: 7/2/02

17

16.13 Figure 1: Image of the RapidChek lateral flow device for E. coli O157 (including H7).

(A) (B) (C) Three RapidChek devices are shown, (a) one which has not be used; (b) one which has been run with a negative meat sample, displaying one red line indicative of a negative result; (c) one device which has been run with a positive meat sample, displaying two red lines indicative of a positive result.

Version 1: 7/2/02

18

Table 1 : E. coli O157 isolates source list Organism Serotype Code Designation Source 1 E. coli O157:H7 ATCCa #35150 Clinical isolate 2 E. coli O157:H7 ATCC #51658 Clinical isolate 3 E. coli O157:NM ATCC #700377 Human feces 4 E. coli O157:H7 ATCC #51659 Unknown 5 E. coli O157:H7 ATCC #700531 Unknown 6 E. coli O157:H7 ATCC # 700728 Unknown 7 8

E. coli E. coli

O157:H7 O157:H7

ATCC # 43894 ATCC #51657

Unknown Unknown

9 E. coli O157:H7 ATCC # 43889 Unknown 10 11

E. coli E. coli

O157:H7 O157:H7

ATCC # 43890 ATCC # 43895

Unknown Food isolate

12 E. coli O157:H7 USDAb 380-94 CDC Clinical isolate 13 E. coli O157:H7 USDA 45753-35 Beef kidney isolate 14 E. coli O157:H7 USDA 505B Food Research Beef isolate 15 E. coli O157:H7 USDA A91124-1 CDC Clinical isolate 16 E. coli O157:H7 USDA C7927 Apple cider outbreak isolate 17 E. coli O157:H7 USDA 413-91 Ground beef survey isolate 18 E. coli O157:H7 USDA Ent C940 Jack in the Box patient isolate 19 E. coli O157:H7 USDA 45750 FSIS meat isolate 20 E. coli O157:H7 USDA 3417-85 CDC Food outbreak isolate 21 E. coli O157:H7 USDA 86-24 Food isolate 22 E. coli O157:H7 USDA A8959-C7 Unknown 23 E. coli O157:H7 PSUc 88.1020 Rabbit isolate 24 E. coli O157:H7 PSU 89.0511 Human isolate 25 E. coli O157:H7 PSU 96.0112 Human isolate 26 E. coli O157:H7 PSU 95.1282 Human isolate 27 E. coli O157:H7 PSU 93.0054 Ground beef 28 E. coli O157:H7 PSU 96.0932 Cow isolate 29 E. coli O157:H7 PSU 88.1448 Cow isolate 30 E. coli O157:H7 PSU 95.1278 Human isolate 31 E. coli O157:H7 PSU 96.0109 Human isolate 32 E. coli O157:H7 PSU 96.0940 Cow isolate 33 E. coli O157:H7 PSU 88.1022 Rabbit isolate 34 E. coli O157:H7 PSU 96.0939 Cow isolate 35 E. coli O157:H7 PSU 96.0428 Cow isolate 36 E. coli O157:H7 PSU 93.0138 Ground beef 37 E. coli O157:H7 R & Fd # 219 Apple cider 38 E. coli O157:H7 R & F # 220 Food isolate 39 E. coli O157:H7 R & F # 221 Beef isolate 40 E. coli O157:NM R & Fe # 244 Clinical, Iowa 41 E. coli O157:H7 R & F # 246 Clinical, Iowa 42 E. coli O157:H7 R & F # 248 Clinical, Iowa 43 E. coli O157:H7 R & F # 249 Clinical, Iowa 44 E. coli O157:H7 R & F #234 Clinical, Iowa 45 E. coli O157:H7 R & F #252 Apple cider 46 E. coli O157:H7 R & F # 377 Ground beef 47 48

E. coli E. coli

O157:H7 O157:H7

R & F # 380 R & F # 383

Beef Beef

49 E. coli O157:H7 R & F # 401 Beef 50 E. coli O157:H7 SLRf 1427 Food isolate AAmerican Type Culture Collection, Manassas, VA; bUnited States Department of Agriculture, Wyndmoor, PA; CPennsylvania State University E. coli Reference Center, University Park, PA; dR & F Laboratories, West Chicago, IL; ER & F Laboratories, isolates from Iowa Department of Health; fSLR, Silliker Laboratories, Inc., South Holland, IL.

Version 1: 7/2/02

19

Table 2 : Non- E. coli O157 isolates Organism Code designation E. coli O86:K1 ATCC # 12701 E. coli O111 ATCC # 33780 E. coli O26:K60

E. coli O91:NM E. coli O142:K86:H6

ATCC # 12795 ATCC # 23980 ATCC # 23985

E. coli O91:H21 ATCC # 51435 E. coli O55:K59 ATCC # 12014 Citrobacter freundi ATCC # 8090 Citrobacter sedlaki

Citrobacter braakii Citrobacter youngae

ATCC # 51115 ATCC # 51113 ATCC # 11102

Klebsiella pneumoniae ATCC # 13883 Proteus vulgaris ATCC # 8427 Proteus mirabilis

Enterobacter cloacae ATCC # 4630 ATCC # 13047

Enterobacter aerogenes ATCC # 15038 Salmonella enteritidis ATCC # 13076 Salmonella anatum ATCC # 9270 Salmonella hadar ATCC # 51956 Salmonella albany ATCC # 51960 Salmonella kentucky ATCC # 9263 Salmonella heidelberg ATCC # 8326 Salmonella muenchen ATCC # 8388 Salmonella newport ATCC # 6962 Salmonella typhimurium ATCC # 14028 Shigella species ATCC # 2354 Escherichia hermanii ATCC # 33650 Escherichia vulneris ATCC # 33821 Escherichia blattae ATCC # 33430 Escherichia species ATCC # 25922 All strains for inclusivity and exclusivity were typed internally according to the USDA/FSIS confirmatory procedure (10).

Version 1: 7/2/02

20

Table 3 – Inclusivity and Exclusivity Study

Organism Media type Total number of strains tested on

RapidChek

Total positive on RapidChek

Sensitivity/ Specificity

(%)

E. coli O157a

RapidChek

50

50

100%

Non E. coli O157b TSB 31 0 100%

AAll E. coli O157 strains were grown up to levels in the range of 108-109 cfu/ml, samples were serially diluted and tested at 106 cfu/ml on the RapidChek device. BAll non E. coli O157 strains were grown to levels in the range of 107-108 and tested neat (without dilutions at this level) on the RapidChek device.

Version 1: 7/2/02

21

Table 4 – External Method Comparison Study for the RapidChek Method with 8 Hour and 18 Hour RapidChek Media and 20 Hour mEC Media Enrichment of Raw Ground Beef

Method Type

(media)

Inoculum Level (cfu/g)

MPN cfu/25g

Total Samples

Examined

Method Positive

Confirmed Positive

Accuracy (%)

Method Agreementa

(%) Rapid✔

(8 h Rapid✔Media)

Non-inoculated (0)

Low (1-10)

High (10-50)

0

2.3

10.7

5

20

20

0

19

20

N/A

19

20

100

100

100

100

100

100

Rapid✔

(18 h Rapid✔Media)

Non-inoculated

(0)

Low (1-10)

High (10-50)

0

2.3

10.7

5

20

20

0

20

20

N/A

20

20

100

100

100

100

95

100

Rapid✔

(mEC Media)

Non-inoculated

(0)

Low (1-10)

High (10-50)

0

2.3

10.7

5

20

20

0

19

20

N/A

19

20

100

100

100

100

100

100

USDA/FSIS

Reference Method (mEC Media)

Non-inoculated

(0)

Low (1-10)

High (10-50)

0

2.3

10.7

5

20

20

0

19

20

N/A

19

20

100

100

100

a Individual RapidChek results to be compared with reference method results Note: background total microbial aerobic count equal 110 cfu/g

Table 5 - Internal Method Comparison Study for the RapidChek Method with 8 Hour (Shaken and Static Cultures) and 18 Hour RapidChek Media and 20 Hour mEC Media Enrichments of Raw Boneless Beef

Method Type

(media)

Inoculum Level(a,b,c)

(cfu/25g)

Total Samples Examine

Method Positive

Confirmed Positive

Accuracy (%)

Method Agreementd

(%) Rapid✔

(8 h Rapid✔Media

Shaken)

Non-inoculated (0)

Low

(1-0.10)

High (10-50)

5

20

20

0

20

20

N/A

20

20

100

100

100

100

40

65 Rapid✔

(8 h Rapid✔Media

Static)

Non-inoculated (0)

Low

(1-0.10)

High (10-50)

5

20

20

0

20

20

N/A

20

20

100

100

100

100

40

65 Rapid✔

(18 h Rapid✔Media)

Non-inoculated (0)

Low

(1-0.10)

High (10-50)

5

20

20

0

19

20

N/A

19

20

100

100

100

100

45

65 Rapid✔

(mEC Media)

Non-inoculated (0)

Low

(1-0.10)

High (10-50)

5

20

20

0

5

15

N/A

5

15

100

100

100

100

85

90 USDA/FSIS Reference Method

(mEC Media)

Non-inoculated (0)

Low

(1-0.10)

High (10-50)

5

20

20

0

8

13

N/A

8

13

100

100

100

a) MPNs for Low and High inoculum were 0.4 and 23.3 cfu/25g respectively b) Pre-stressed inoculum plate counts for Low and High inoculum equal 5 and 20 cfu/25g respectively. c) Post-stressed inoculum plate counts for Low and High inoculum equaled 2.7 and 10.9 cfu/25g respectively d) Individual RapidChek results to be compared with reference method results Note: background total microbial aerobic count equal 4.1E4 cfu/g

Version 1: 7/2/02

23

Table 6- Internal Method Comparison Study for the RapidChek Method with 8 Hour and 18 Hour RapidChek Media and 20 Hour EEB Media Enrichments of Apple Cider

Method Type

(media)

Inoculum Level(a,b,c)

(cfu/25ml)

Total Samples

Examined

Method Positive

Confirmed Positive

Accuracy (%)

Method Agreementd

(%)

Rapid✔ (8 h

Rapid✔Media)

Non-

inoculated (0)

Low

(1-0.10)

High (10-50)

5

20

20

0

16

20

N/A

16

20

100

100

100

100

95

100

Rapid✔

(18 h Rapid✔Media)

Non-

inoculated (0)

Low

(1-0.10)

High (10-50)

5

20

20

0

20

20

N/A

20

20

100

100

100

100

85

100

Rapid✔

(EEB Media)

Non-

inoculated (0)

Low

(1-0.10)

High (10-50)

5

20

20

0

20

20

N/A

20

20

100

100

100

100

85

100

FDA/BAM Reference Method

(EEB Media)

Non-

inoculated (0)

Low

(1-0.10)

High (10-50)

5

20

20

0

17

20

N/A

17

20

100

100

100

e) MPNs for Low and High inoculum were 2.4 and 10.8 cfu/25mL respectively f) Pre-stressed inoculum plate counts for Low and High inoculum equal 5 and 20 cfu/25mL respectively. g) Post-stressed inoculum plate counts for Low and High inoculum equaled 4.1 and 17 cfu/25mL respectively h) Individual RapidChek results to be compared with reference method results Note: background total microbial aerobic count equal 10cfu/mL

Version 1: 7/2/02

24

Table 7- Temperature Latitude Study/Reading Time Study

Inoculum Level of E. coli O157

Number of

Samples tested

Reading Time of

Device (minutes)

Number of RapidChek Positive Results at Different Temperatures/Total Tested

12°°°°C 20°°°°C 30°°°°C

0 5 2 0/5 0/5 0/5 (negative) 4 0/5 0/5 0/5

6 0/5 0/5 0/5 8 0/5 0/5 0/5 10 0/5 0/5 0/5 12 0/5 0/5 0/5 14 0/5 0/5 0/5 16 0/5 0/5 0/5 18 0/5 0/5 0/5 20 0/5 0/5 0/5

1-10 cfu/25g

5 2 2/5 2/5 4/5

(positive) 4 5/5 5/5 5/5 6 5/5 5/5 5/5 8 5/5 5/5 5/5 10 5/5 5/5 5/5 12 5/5 5/5 5/5 14 5/5 5/5 5/5 16 5/5 5/5 5/5 18 5/5 5/5 5/5 20 5/5 5/5 5/5

Version 1: 7/2/02

25

Table 8: Sample Enrichment Time Latitude Study

Inoculum level

Sample Number

tested

Total number of RapidChek Positive Samples/Total Tested

Accuracy (%)

Rapid✔Media (8 h)

Non-inoculated

(0 cfu/ 25g)

2

7 h

0/2

8 h

0/2

9 h

0/2

100

Low (1-5 cfu/ 25g)

5

5/5 5/5 5/5 100

High (10-25 cfu/25g)

5 5/5 5/5 5/5 100

Rapid✔Media

(18 h)

Non-inoculated (0 cfu/ 25g)

2

17 h

0/2

18 h

0/2

19 h

0/2

100

Low (1-5 cfu/ 25g)

5

5/5 5/5 5/5 100

High (10-25 cfu/25g)

5 5/5 5/5 5/5 100

Table 9: Sample Volume Study

Sample Volume (µµµµl)

RapidChek Positive Results for Inoculated

samples

RapidChek Positive Results for Non-

inoculated Samples

Accuracy (%)

100

2/2

0/2

100

200 2/2 0/2 100 300 2/2 0/2 100 400 2/2 0/2 100 500 2/2 0/2 100

Version 1: 7/2/02

26

Table 10: Lot to Lot Variability of RapidChek Lateral Flow Method RapidChek Lot

# Method

Type (media)

Inoculum Level (cfu/g)

MPN/g Total Samples

Examined

Method Positive

Confirmed Positive

2D1054 Rapid✔

(18 h Rapid✔Media)

Non-

inoculated (0)

Low (0.04-0.4)

High (0.4-2)

0/g

0.004/g

0.24/g

5

20

20

0

19

20

0

19

20

2D1084 Rapid✔

(18 h Rapid✔Media)

Non-

inoculated (0)

Low (0.04-0.4)

High (0.4-2)

0/g

0.004/g

0.24/g

5

20

20

0

19

20

0

19

20

2D1103B Rapid✔

(18 h Rapid✔Media)

Non-

inoculated (0)

Low (0.04-0.4)

High (0.4-2)

0/g

0.004/g

0.24/g

5

20

20

0

19

20

0

19

20

Version 1: 7/2/02

27

Table 11: Accelerated Stability Study

Time (days)

Inoculum level

(cfu/25g)

RapidChek Device Positive Results/ Total after storage At different temperatures

4°°°°C 25°°°°C 37°°°°C 45°°°°C

Accuracy (%)

0

0

0/3

0/3

0/3

0/3

100

Low (1-10) 3/3 3/3 3/3 3/3 100 High (10-50) 3/3 3/3 3/3 3/3 100

21

0

0/3

0/3

0/3

0/3

100 Low (1-10) 3/3 3/3 3/3 3/3 100 High (10-50) 3/3 3/3 3/3 3/3 100

46 0 0/3 0/3 0/3 0/3 100 Low (1-10) 3/3 3/3 3/3 3/3 100 High (10-50) 3/3 3/3 3/3 3/3 100

Table 12: RapidChek Testing of Live Sample and Boiled Samples from 8 Hour SDI RapidChek and 20 mEC Enriched Ground Beef

Enrichment Media

Inoculum Level

Number of Samples Tested

Number of Live Samples

RapidChek Positive

Number of Boiled Samples

RapidChek Positive

% Agreement

8 Hour Rapid✔Media

0 5 0 0 100

Low (0.04-0.12) 10 10 10 100

High (0.12-0.2) 10 10 10 100

20 Hour mEC 0 5 0 0 100

Low (0.04-0.12) 10 9 10 90

High (0.12-0.2) 10 10 10 100

Version 1: 7/2/02

28