ABSTRACT 3M Company, St. Paul, MN, United States: Julie Yang
Transcription
ABSTRACT 3M Company, St. Paul, MN, United States: Julie Yang
P2-05: Performance of the 3M Molecular Detection Assay ™ Salmonella and the 3M Molecular Detection Assay Listeria in Environmental and Primary Production Samples ™ 3M Company, St. Paul, MN, United States: Julie Yang, Cynthia Zook, Micki Rosauer ABSTRACT Introduction: Testing for Salmonella and Listeria is a critical component of food safety programs as infection by these pathogens can result in significant adverse health conditions and economical losses. The 3M™ Molecular Detection Assay Salmonella and the 3M™ Molecular Detection Assay Listeria were developed for the rapid and specific detection of these contaminants in samples after enrichment. Purpose: To evaluate the performance of the new 3M methods in a variety of environmental and primary production samples. conducted: Set 1) where one of the duplicates was enriched blank and one was artificially contaminated with ~10 CFU of the target organism; and Set 2) where single samples were enriched blank to evaluate for native contamination. Enrichments were tested using the 3M Detection Molecular Assays and selective and differential agar and/or quantitative PCR. A Chi square (X2) test was used to compare the results for significant differences. Results: Compared to agar or q-PCR, accuracy, specificity and sensitivity were: 99%, 100% and 95% respectively for the 3M Molecular Detection Assay Salmonella and 95.3%, 100%, 94.9% Methods: More than 550 environmental and primary production samples were collected in duplicates. Two sets of testing were respectively for the 3M Molecular Detection Assay Listeria. No significant differences were observed between the 3M method results and the chromogenic agar results or q-PCR results as indicated by a X2 value of less than 3.84 for either analyte. Significance: The new methods were determined to be reliable and accurate. For all samples evaluated, the 3M Molecular Detection Assay Salmonella and the 3M Molecular Detection Assay Listeria demonstrated comparable results to the other methods for the rapid, automated detection of these organisms. METHODS The 3M Molecular Detection Assays are used with the 3M™ Molecular Detection System for the rapid and specific detection of pathogen in enriched food, feed and food process environmental samples. The 3M Molecular Detection Assays use isothermal amplification of nucleic acid sequences with high specificity, efficiency and rapidity, and bioluminescence to detect the amplification. Presumptive positive results are reported in real-time while negative results are displayed after the assay is completed. 4. F or samples collected with Neutralizing Buffer, a portion of enriched sample was diluted 1:2 into the appropriate type of sterile 3M enrichment broth before testing using the 3M Molecular Detection System. 5. Enrichments were also either streaked onto chromogenic agar plates or tested by q-PCR, as reference points. A Chi-square (X2) test was used to compare the results for significant differences. 6. Calculation and Interpretation: Accuracy, specificity and sensitivity of the 3M Method. 1.Cultures: Target cultures (Table 1) were propagated in 3M™ Buffered Peptone Water ISO (BPW ISO) enrichment broth for ~18 hours at 37°C (Salmonella) or in 3M™ Modified Listeria Recovery Broth (mLRB) for ~24 hours at 37°C (Listeria monocytogenes). To verify spike levels used to inoculate the samples, cultures were enumerated using 3M™ Petrifilm™ Aerobic Count Plates. Table 1. Study Test Strains List Genus, Species/Serotype Salmonella Typhimurium Responses Chromogenic agar and qPCR Positive (R+) Chromogenic agar and qPCR Negative (R–) 3M Method Positive (A+) +/+ Positive Agreement (PA) R– –/+ Positive Deviation (PD) as — A+ 3M Method Negative (A–) A– +/– Negative Deviation (ND) as — R+ ( ) ( ) –/– Negative Agreement (NA) Accuracy (AC), Specificity (SP) and Sensitivity (SE) of the 3M method were calculated as follows (where N=total number of samples): (PA+NA) NA PA AC = x 100% SP = x 100% SE = x 100% N (PD+NA) (PA+ND) ATCC 14028 Salmonella Enteriditis 13076 Salmonella — Environmental and Poultry Rinse Studies Salmonella Newport Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes 6962 7644 19118 43256 142 different environmental samples, including poultry drag swabs, were collected in duplicate from various farms. Surfaces tested included concrete floors and ceiling, wooden roosts, metal feeders, and stainless steel equipment and pipes. 84 bird rinses were provided by 9 different poultry processing plants. Table 2 indicates matrices, sampling devices and enrichment volume of 3M BPW ISO used. ATCC — American Type Culture Collection 2. Sample enrichments were performed for Salmonella in 3M BPW ISO at 37°C and Listeria in 3M mLRB at 37°C. All enrichment broths were pre-warmed to the incubation temperature prior use. 3. 3M Molecular Detection Assay: Each enrichment was extracted and an aliquot of the lysate was tested using the 3M Molecular Detection System (Figure 1). 3M Method Listeria: Sample enrichment Incubation (24 –28 hours) 3M BPW ISO Enrichment Volume 30mL 3M™ Dry-Sponge Surfaces 10mL D/E Neutralizing broth 50mL 3M™ Dry-Sponge with String Poultry fecal drag swab 10mL BPW 50mL Listeria — Environmental Samples Studies LS 431 environmental samples were collected in duplicates from dairy farms, cattle farms and a ready-to-eat (RTE) food processing plant, where sampling locations were chosen specifically to increase the likelihood of finding naturally-occurring Listeria. Table 3 summarizes surfaces tested, sampling devices and enrichment volume of 3M mLRB used. Transfer 20µL enriched sample to lysis tube. Table 3. Surfaces and Collection Devices Summary Sampling Devices Surface Material 3M™ Dry-Sponge Stainless steel 0-20ºC 99-101ºC Cool 10 minutes on chill block. Let sit 5 minutes. Heat 15 minutes at 100°C (±1°) 20µL Transfer 20µL lystate to reagent tubes containing lyophilized pellet. Place Speed Loader Tray into instrument. Close the lid and start run. Hydrating Solution BPW Two sample sets were tested as follows (All enrichments were performed at 37°C for 18 hours): Set 1: Suspensions of Salmonella Typhimurium, S. Enteritidis or S. Newport were used to artificially contaminate one of the duplicates with ~10 CFU. The other duplicate was enriched blank. Set 2: Single samples enriched as blank to evaluate for native contaminations. Figure 1. Simplified Protocol for Productivity 3M Method Salmonella: Sample enrichment Incubation (18 –24 hours) Table 2. Matrices and Collection Devices Summary Sampling Devices Sample Type None Poultry carcass rinse Transfer closed tubes to Speed Loader Tray. Performs amplicifations in 75 minutes. Automated and color coded real-time results. TTR: Neutralizing or Hydrating Solution D/E Neutralizing broth 3M mLRB Enrichment Volume 225mL 3M™ Dry-Sponge Concrete D/E Neutralizing broth 225mL 3M™ Dry-Sponge 3M™ Sponge-Stick 3M™ Quick Swab 3M™ Enviro Swab 3M™ Enviro Swab Plastic Various surfaces Metal drains Plastic piping Various surfaces Neutralizing buffer D/E Neutralizing broth Letheen broth Modified Letheen broth Modified Letheen broth 225mL 225mL 10mL 10mL 10mL Three sample sets were tested as follows: Set 1: Paired samples were incubated blank, or were spiked with suspensions of L. monocytogenes at a level of ~10 CFU per sponge, then incubated for 22 hours at 37°C. Set 2: Paired samples were incubated blank, or were spiked with suspensions of L. monocytogenes at a level of ~10 CFU per sponge, then incubated for 24 hours at 37°C. Set 3: Single samples collected from a RTE food processing plant were enriched blank for 22, 26 and 28 hours at 37°C to evaluate for native contamination. 20 –30 hours RESULTS CONCLUSIONS No significant differences were observed between the 3M method results and the chromogenic agar results or q-PCR results as indicated by a X2 value of less than 3.84 for either analyte (Table 4). A Chi square (X2) of ≥ 3.84 indicates that the proportions positive for the different methods differ significantly at p ≤ 0.05. This criterion is used by AOAC International for analysis of confirmed positives; here it used for analysis of presumptive positive results. The new 3M Molecular Salmonella and Listeria spp. methods were determined to be reliable and accurate, and demonstrated comparable results to chromogenic agar and/or q-PCR results. The 3M methods were shown to be compatible with a variety of environmental samples, including poultry drag swabs, bird rinses and fecal containing samples. In addition, the process flow was more streamlined than the q-PCR method and faster than the cultural methods. Table 4. Performance Results Summary Target Type of Sample Salmonella Environmental Salmonella Bird Rinse Listeria Environmental n 142 84 431* Accuracy 99% 97.6% 95.3% Specificity 100% 100% 100% Sensitivity 95% 93.9% 94.5% *All samples combined (n = 431, incubation time 22, 24, 26 or 28 hours) References a. Feldsine, Abeyta and Andrews, 2002. Section 5.3.1 Test for Significant Difference (X2), Journal of AOAC, Vol. 85, No. 5, P. 1187-1200. X2 0.00 1.33 1.8 ACKNOWLEDGEMENTS The authors thank Molly Rother, Amber Turner and Olivia Trittipo for their dedicated laboratory contributions. © 3M 2012. 3M and Petrifilm are trademarks of 3M.