Microbiology Methods for ERP Review 3-2020

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GENE-UP SLM Matrix Extension

June 19, 2017

proximity from hybridizing to the template DNA. The resulting fluorescent signal from the FRET interaction, 1 which forms a real ‐ time amplification curve, is how the amplified target is detected by the GENE ‐ UP 2 Thermocycler. After the PCR cycling program finishes, the PCR product(s) are melted to determine the 3 presence of the target DNA. The software uses both the real ‐ time amplification curve and the melt peak to 4 make a positive or negative call. 7 The GENE ‐ UP Salmonella method was originally validated by Idaho Technology as the R.A.P.I.D. LT Food 8 Security System (FSS). Three food matrices, cooked ham, raw chicken and chocolate (all 25 g) were 9 evaluated. The GENE ‐ UP Salmonella uses the same technology and reagent formulation in a 96 well 10 automated format. In 2015, a modification study was performed using the GENE ‐ UP instrument to extend 11 the matrix claim and add Buffered Peptone Water (BPW) as the enrichment broth. The matrices added 12 included raw ground beef (25 and 375 g), raw chicken breast (25 g), raw fish (25 g), creamy peanut butter 13 (25 g), vanilla ice cream (25 g), dry pet food (25 g) and stainless steel environmental sponge samples. An 14 inclusivity and exclusivity study was also conducted in BPW, with all inclusivity isolates were correctly 15 detected, and all exclusivity isolates were correctly undetected. 16 17 This validation study to extend the matrix claim further and add two new enrichment media was conducted 18 under the AOAC Research Institute Performance Tested Method SM program and the AOAC INTERNATIONAL 19 Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental 20 Surfaces (4). Method developer studies were conducted in the laboratories of bioMérieux (St. Louis, MO), 21 and included the inclusivity/exclusivity and matrix studies for all claimed matrixes. The Independent 22 laboratory study was conducted by Q Laboratories (Cincinnati, OH), and included a matrix study for five of 23 the claimed matrixes. 26 The inclusivity/exclusivity evaluation consisted of analyzing 101 Salmonella strains, the FDA SAFE list, (Table 27 1) and 53 closely related non ‐ target isolates (Table 2) using modified Tryptic Soy Broth (mTSB) and Buffered 28 Peptone Water with potassium sulfite (BPW + 0.5% K 2 SO 3 ) as the enrichment media for the GENE UP 29 Salmonella method. All exclusivity organisms were cultured in brain heart infusion (BHI) for 24 h at 30 conditions that would promote optimal growth. All Salmonella isolates were cultured in mTSB (42 ± 1°C for 31 18 h) and BPW + 0.5% K 2 SO 3 (35 ± 1°C for 18 h). Following incubation, Salmonella cultures were diluted to 32 100 x the limit of detection (LOD) while exclusivity cultures were analyzed without dilution. After 33 incubation, inclusivity and exclusivity test cultures were randomized, blind coded and analyzed according to 34 the GENE ‐ UP Salmonella method. 35 36 Of the 101 Salmonella strains evaluated, 101 target strains were detected. Of the 53 non ‐ Salmonella strains 37 evaluated, 53 were not detected (Tables 1 and 2). 40 Thirteen matrixes were evaluated in this study (Table 2). A background screen of each food matrix was 41 conducted prior to inoculation and no natural contamination of the target organisms was detected in the 42 test matrices (5). Each matrix was inoculated with a different strain of Salmonella as specified in Table 3. 43 Each inoculum was prepared by transferring a pure isolated colony of the specified organism from 44 trypticase soy agar with 5% sheep’s blood (SBA) into BHI broth and incubating the inoculum at 35 ± 2°C for 45 24 ± 2 h. Post incubation, the inoculum was diluted using BHI, added to a bulk lot of test material and 46 homogenized. The inoculum for the dry products was suspended in 10% non ‐ fat dry milk and lyophilized 47 prior to inoculation. For heat processed products, the organisms were heat stressed by incubating the 48 culture for 10 min at 50 ± 1°C prior to inoculation. The heat stressed culture was plated onto a selective 49 5 6 Validation Study 24 25 Inclusivity and Exclusivity 38 39 Matrix Study

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