Result: COMPLETE Report Date: June 12, 2017

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1 Send to: Dr. Preetha Biswas Neogen Corporation 620 Lancaster Place Lansing, MI Result: COMPLETE Report Date: June 12, 2017 Customer Name: Neogen Corporation Location of Testing: NSF Ann Arbor Description: Listeria Right Now-Validation Study Test Type: Test Only Job Number: J Project Number: NSF Corporate: C Project Manager: K. Martin Executive Summary: Neogen Corporation requested NSF International to perform a validation study to evaluate the performance of the Listeria Right Now Assay (LRN) for detection of Listeria spp. In environmental swabs without enrichment. Thank you for having your product tested by NSF International. Please contact your Project Manager if you have any questions or concerns pertaining to this report. Report Authorization: Jesse Miller Director, Applied Research Center FI J Page 1 of 28

2 Experimental Summary: Target microorganisms: Listeria monocytogenes ATCC Method Development: To determine the most suitable method for culture preparation a comparison of bacterial titers between the uses of a McFarland standard and historical expected titer. 1. A portion of a h overnight culture of L. monocytogenes was washed by centrifugation and re-suspended in sterile 0.85% saline solution to the equivalent of a 4.0 McFarland standard. 2. Spread plate densities were then taken of the 4.0 McFarland Suspension and compared to the overnight culture alone. 3. NSF determined that the concentration of the L. monocytogenes test strain is 5 times lower than the turbidity standard that the provided product literature suggests. The lab adjusted the spike densities accordingly to more accurately meet target spike densities specified by the client. Environmental Surface Study: Cleaning: Stainless steel surfaces were cleaned according to the following procedure before testing: 1. Clean the stainless steel surface with alkaline detergent (or 1N NaOH), by soaking Kimwipes with detergent and scrubbing (wipe forcefully) the surface and then rinsing with DI water. 2. Spray 10% fresh bleach (prepared within 24 h of use) on the surface, wait for 10 min, then scrub and Kimwipe. 3. Rinse the surface with deionized water and allow to air dry. 4. Spray 70% isopropyl alcohol on the surface, and wait for 5minutes. 5. Rinse with sterile deionized water and allow to air dry. Culture Preparation: All culture preparation was conducted using the following method: 1. Observe the table below for the inoculum size and number of replicates. Listeria monocytogenes (CFU/mL) Organisms Negative (Positive) (Level 1) 1x (Level 2) 24 (Level 3) 60 Total (Excluding Negative) L. monocytogenes Background* *Background organisms: A cocktail of Pseudomonas aeruginosa, Bacillus subtilis, and Enterococcus faecium at 1x10 4 CFU/mL FI J Page 2 of 28

3 2. L. monocytogenes Prep a. Centrifuge a h overnight culture of Lm at 3500Xg for 15 minutes and suspend in 1 ml 0.85% saline solution b. Prepare a 4.0 McFarland Standard in 0.85% saline solution from the centrifuged stock culture. i. Theoretical concentration: 1.2x10 9 CFU/mL. c. Dilute 4.0 McFarland solution to 1x10 5, 600, 240 and 80 CFU/mL in BPW. i. Create the 1x10 5 CFU/mL stock with a final volume of 10mL. ii. Pipette 1mL of the 4.0 McFarland in 9mL BPW, to make iii. Pipette 1mL of the 10-1 in 9mL BPW, to make iv. Pipet 417µL of 10-2 in 9.6mL BPW, to make 1x10 5 CFU/mL. d. Create the 600 CFU/mL stock with a final volume of 40 ml. i. Pipet 0.1mL of the 4.0 McFarland in 9.9mL BPW, to make ii. Pipette 0.1mL of the 10-2 in 9.9mL BPW, to make iii. Pipette 1mL of 10-4 in 39mL BPW, to make 600 CFU/mL. e. Create the 240 CFU/mL stock by combining. i. Pipette 4mL of 600 CFU/mL in 6mL BPW, to make 240 CFU/mL. f. Create the 80 CFU/mL stock by combining the following: i. Pipette ml of 600 CFU/mL in 8.7mL BPW, to make 80 CFU/mL. g. Add 1 ml of each diluted concentration of L. mono (1x10 5, 600, 240 and 80 CFU/mL) respectively, to 4 tubes containing 9 ml BPW. h. The resulting volume will be 10 ml with the following final concentrations: i. L. mono: 1x10 4, 8, 24 and 60 CFU/mL in BPW 3. L. monocytogenes + Background Prep a. Centrifuge a h overnight cultures of Pa 10145, Bs 9372, and Ef at 3500Xg for 15 minutes and suspend in 1 ml 0.85% saline solution. b. Prepare a 1.0 McFarland Standard of each organism in 0.85% saline solution from the centrifuged stock culture. i. Theoretical concentration: 3.0x10 8 CFU/mL. c. Dilute each 1.0 McFarland standard solution 1:1000 in BPW. i. Theoretical concentration 3.0x10 5 CFU/mL. d. Pool all organisms into a single tube. e. To 4 tubes of 8 ml BPW, add 1 ml of the organism pool. The resulting volume will be 9 ml. f. Add 1 ml of each diluted concentration of L. mono (1x10 5, 600, 240 and 80 CFU/mL) respectively, to the 4 tubes containing the 9 ml diluted background organism cocktail. g. The resulting volume will be 10 ml with the following final concentrations: i. L. mono: 1x10 4, 60, 24 and 8 CFU/mL in BPW. ii. Background: 1.0x10 4 CFU/mL in BPW. FI J Page 3 of 28

4 4. Inoculum count: Once the spike culture has been prepared, perform spread plating of the spike. Observe the table below for dilutions and replicates to plate. Perform the plating as follows: a. Right after preparing the inoculum (pre-spike), and b. After spiking all the surfaces (post-spike) Inoculum Replicate Solutions Organisms Dilution count plates/dilution L. monocytogenes 10-6, Background org , McFarland Standard Background org , Background org , Pre-spike Pooled Background 10-3, CFU/mL Inoculum L. monocytogenes 0.25 ml 3 60 CFU/mL Inoculum + 1.0x10 4 CFU/mL background Total 10-2, cocktail 60 CFU/mL Inoculum L. monocytogenes 0.25 ml 3 Post-spike 60 CFU/mL Inoculum + 1.0x10 4 CFU/mL background Total 10-2, cocktail 5. An aliquot of 0.25mL of the inoculum is applied on an area of 4 x 4 ± 0.5 per surface and spread using a sterile spreader. 6. Spike 2 square surfaces and spread using a sterile spreader. Wait for 1 minute. Spike the next 2 square surfaces. a. If there are no squares with 50% dried inoculum, repeat this spiking pattern for the next pair of squares. Processing: 1. Spiking, quality control and processing was carried out in accordance with Appendix B: Listeria Right Now Assay- Validation Study Plan for Independent Laboratory. Additional testing parameters: All test swabs were stored at 4 o C for a minimum of 30 minutes before testing with the ANSR assay. FI J Page 4 of 28

5 Summary/Conclusion The purpose of this study was to evaluate the performance of the Listeria Right Now (LRN) assay for the detection of Listeria spp. in environmental swabs without a prior enrichment process. The surfaces of 4 x 4 squares of food-grade stainless steel were inoculated with different levels of Listeria monocytogenes and a consortium of competing organisms, including Pseudomonas aeruginosa, Bacillus subtilis, and Enterococcus faecium. After allowing the inoculum to partially dry (50%), surface samples were collected using semi-paired swabs. One swab was tested by the Listeria Right Now assay and the other swab was enriched by the culture method. The swab for the culture method was enriched overnight at 37 C in growth medium and an aliquot plated on to agar plates for detection on the following day. In the Listeria Right Now test, the entire collected contents of the swab were subjected to sample processing and testing on the same day. After expression of the swab in the lysis buffer, one-half of the volume (0.5 ml) was taken for the lysis incubation steps. Next, a portion of the lysed sample was transferred to a strip tube containing lyophilized reaction reagents. The tubes were sealed and incubated at 56 C on the Neogen reader. Results generated by the reader were displayed in the LRN software. No false negatives, false positives or invalids were observed during this study. The evaluation determined that under the conditions employed in this study, the enrichment-free Listeria Right Now method is as sensitive as the enrichment-based culture reference method for detection of L. monocytogenes on a stainless steel surface. Test Notes: Additional inoculum counts were taken during surface testing to adequately examine bacterial titers during and after spiking test surfaces. MOX agar was outside of the ph range of 7.0 +/ Quality control was performed on the agar batch and it passed. The McFarland suspensions were first prepared in 0.85% sterile saline solution before being diluted in BPW. This was due to coloration present in the BPW that would have affected the preparation of the McFarland suspension. References: Neogen ANSR User Manual Scope of Work Revisions: Scope of work authorized on March 7, Version authorized on April 5, 2017 contained the following revisions: o Updated costs associated to revised scope of work o Updated timeline associated to revised scope of work o Updated Appendix B protocol per edits made by Bryan Schindler on 4/4/2017 o Updated footer to include page numbering new (project) version number FI J Page 5 of 28

6 Results FI J Page 6 of 28

7 Table 1: Method development results to determine the most suitable method for culture preparation by comparing bacterial titers between the uses of a McFarland standard and historical expected titer. TNTC= too numerous to count. Round One Inoculum Prep type Theoretical CFU/Plate Actual CFU/Plate McFarland Standard TNTC 15 Historical Density 201 Round Two Inoculum Prep type Theoretical CFU/Plate Actual CFU/Plate McFarland Standard 3 15 Historical Density 46 Round Three Inoculum Prep type Theoretical CFU/Plate Actual CFU/Plate McFarland Standard 1 15 McFarland Standard Historical Density 8 Table 2: Environmental surface study inoculum counts. Inoculum Count CFU/mL L. monocytogenes (McFarland std) 4.60E+08 P. aeruginosa (McFarland std) 1.20E+09 B. subtilis (McFarland std) 2.50E+07 E. faecium (McFarland std) 3.14E+09 Background Cocktail 10X stock 5.10E+04 Inoculum Count (pre-spike) CFU/mL L. monocytogenes (target 60 CFU/mL) 34 L. monocytogenes (target 60 CFU/mL) + Background 1.00E+03 Inoculum Count (post-spike) CFU/mL L. monocytogenes (target 60 CFU/mL) 56 L. monocytogenes (target 60 CFU/mL) + Background 7.10E+03 *Possible lab accident occurred during the enumeration of L. monocytogenes (target 60 CFU/mL) + Background. **The concentration of the Background Cocktail in the inoculum suspension was 5.10E+03 CFU/mL; 10-fold less than the Background Cocktail 10X stock (5.10E+04 CFU/mL). FI J Page 7 of 28

8 Table 3: Environmental surface study results for Listeria monocytogenes and background organisms. Listeria monocytogenes +Background Organisms Swab 1 Swab 2 Swab 1 Swab 2 LRN If Retest MOX Conf LRN If Retest MOX Conf Negative Positive: 4.8E+4 CFU/coupon (Theoretical 2.4E+4 CFU/swab) 1 Negative N/A Negative N/A 1 Positive N/A Positive Positive 2 Negative N/A Negative N/A 2 Positive N/A Positive Positive 3 Negative N/A Negative N/A 3 Positive N/A Positive Positive 4 Negative N/A Negative N/A 4 Positive N/A Positive Positive 5 Negative N/A Negative N/A 5 Positive N/A Positive Positive Level 1: 6 CFU/coupon Level 2: 18 CFU/coupon (Theoretical 3 CFU/swab) (Theoretical 9 CFU/swab) 1 Positive N/A Negative N/A 1 Positive N/A Positive Positive 2 Positive N/A Positive Positive 2 Positive N/A Positive Positive 3 Positive N/A Positive Positive 3 Positive N/A Positive Positive 4 Positive N/A Positive Positive 4 Positive N/A Positive Positive 5 Positive N/A Positive Positive 5 Positive N/A Positive Positive 6 Positive N/A Positive Positive 6 Positive N/A Positive Positive 7 Positive N/A Negative N/A 7 Positive N/A Positive Positive 8 Positive N/A Negative N/A 8 Positive N/A Positive Positive 9 Positive N/A Positive Positive 9 Positive N/A Positive Positive 10 Positive N/A Negative N/A 10 Positive N/A Positive Positive 11 Negative N/A Positive Positive 11 Positive N/A Positive Positive 12 Positive N/A Positive Positive 12 Positive N/A Positive Positive 13 Positive N/A Negative N/A 13 Positive N/A Positive Positive 14 Positive N/A Positive Positive 14 Positive N/A Positive Positive 15 Positive N/A Negative N/A 15 Positive N/A Positive Positive Level 3: 45 CFU/coupon (Theoretical 22.5 CFU/swab) 1 Positive N/A Positive Positive 2 Positive N/A Positive Positive 3 Positive N/A Positive Positive 4 Positive N/A Positive Positive 5 Positive N/A Positive Positive 6 Positive N/A Positive Positive 7 Positive N/A Positive Positive 8 Positive N/A Positive Positive 9 Positive N/A Positive Positive 10 Positive N/A Positive Positive 11 Positive N/A Positive Positive 12 Positive N/A Positive Positive 13 Positive N/A Positive Positive 14 Positive N/A Positive Positive 15 Positive N/A Positive Positive Note: Inoculum spikes for levels 1, 2, and 3 contained 1.5E+03 CFU per coupon (theoretical 7.6E+02 CFU per swab) of background cocktail. FI J Page 8 of 28

9 Table 4: Environmental surface study results for Listeria monocytogenes and background organisms on stainless steel. Level Theoretical Inoculum (CFU/swab) Sample Number LRN Positive % LRN Positive Culture Positive % Culture Positive Negative % 0 0% Positive 2.4E % 5 100% L % 9 60% L % % L % % Note: Table 4 presents the results for the environmental surface study using a challenge inoculum of L. monocytogenes plus a consortium of competing organisms. Three different inoculation levels were evaluated on the stainless steel carriers: Level 1 = 3 CFU, Level 2 = 9 CFU and Level 3 = 22.5 CFU (theoretical CFU/swab). At Level 1, the detection rates for LRN and the reference enrichment-based culture method were 93% and 60%, respectively. At Levels 2 and 3, the detection rates for LRN and the reference enrichment-based culture method were 100%. No false negatives, false positives or invalids were observed during this study. The data illustrates that under the conditions employed in this study Listeria Right Now is as sensitive as the enrichment-based culture reference method for detection of L. monocytogenes on a stainless steel surface. FI J Page 9 of 28

10 Appendix A Listeria Right Now Assay-Validation Study Plan for Independent Laboratory FI J Page 10 of 28

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