SureTect Salmonella species PCR Assay AOAC-RI PTM Validation: Inclusivity and Exclusivity

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1 SureTect Salmonella species PCR Assay AOAC-RI PTM Validation: Inclusivity and Exclusivity Study Report Jonathan Cloke, Thermo Fisher Scientific, Wade Road, Basingstoke, Hampshire, RG24 8PW, UK Inclusivity and Exclusivity As part of the validation study for AOAC Research Institute (RI) Performance Tested Methods SM validation, the inclusivity and exclusivity of the Thermo Scientific SureTect Salmonella species PCR Assay (PT0100A), a new real-time PCR assay for the detection of Salmonella in food and environmental samples was evaluated. Results showed that the SureTect Salmonella species Assay was equivalent in performance to the ISO reference method (ISO 6579:2002). The following is a summary of the inclusivity and exclusivity part of the study. Methodology Choice of strains: One hundred and seventeen inclusivity isolates covering all species and sub-species of Salmonella and thirty-six exclusivity isolates were analyzed as part of the AOAC-RI PTM study. All isolates used in this study were selected from the culture collections at Thermo Fisher Scientific, Basingstoke, UK and originated from either national culture collections or food microbiology laboratories. All testing was conducted according to AOAC-RI guidelines and the study protocol, which was reviewed and approved by AOAC-RI. Culture enrichment: For each inclusivity isolate, one colony was inoculated into a tube containing Buffered Peptone Water (ISO) (BPW (ISO)) and incubated for 18 to 24 hours at 37 C. The broth was then diluted with Maximum Recovery Diluent (MRD) to dilute the target cultures to a level of approximately 10 4 CFU/ml before analyzing with the SureTect protocol. Each exclusivity isolate was inoculated into 10ml Tryptone Soya Broth and incubated for 18 to 24 hours at 37 C. Prepared cultures were analyzed with the SureTect Assay, at the growth level achieved during the incubation time, without being diluted. Method: 10µl of SureTect Proteinase K Reagent was added to each of the required SureTect Lysis Tubes (prefilled with Lysis Reagent 1). 10µl of the sample prepared as detailed above was added to the Lysis Tubes. The SureTect Lysis Tubes were then heated at 37 C for 10 minutes, followed by 95 C for 5 minutes. The tubes were then cooled for 5 minutes and 20µl aliquots of the lysates were transferred to SureTect PCR Tubes containing PCR tablets that were then immediately transferred to the Thermo Scientific PikoReal Real-Time PCR Instrument for processing. Results: Inclusivity and exclusivity results are summarized in tables one and two respectively. All 117 inclusivity isolates tested returned positive results and all of the exclusivity isolates gave negative results with the SureTect System.

2 2 Salmonella Aberdeen TCC 1612 Salmonella Abortusequi TCC 2296 Salmonella Adelaide OCC 2536 Salmonella Agona TCC 1619 Salmonella Alabama TCC 1621 Salmonella Allerton TCC 1623 Salmonella Amherstiana TCC 1624 Salmonella Amsterdam TCC 1625 Salmonella Anatum TCC 1626 Salmonella Binza TCC 2027 Salmonella bongori TCC 2274 Salmonella bongori TCC 2275 Salmonella bongori TCC 2402 Salmonella bongori TCC 2398 Salmonella Bovis-Morbificans TCC 2001 Salmonella Braenderup TCC 1630 Salmonella Brandenberg TCC 1998 Salmonella Bredeney TCC 2019 Salmonella Breukelen TCC 2287 Salmonella Cambridge TCC 1632 Salmonella Cerro TCC 2016 Salmonella Cholerasuis TCC 1852 Salmonella Colombo TCC 2031 Salmonella Corvallis TCC 1633 Salmonella Dahlem TCC 2036 Salmonella Derby TCC 2008 Salmonella Dublin TCC 802 Salmonella Dusseldorf TCC 1634 Salmonella Ealing TCC 1999 Salmonella Eastbourne TCC 2023 Salmonella Emek TCC 1692 arizoniae TCC 1995 arizoniae TCC 2267 arizoniae TCC 2268 Eilbek TCC 2034 TCC 2035 TCC 1997 TCC 2276 houtenae TCC 2032 houtenae TCC 2033 houtenae Seminole houtenae Wassenaar TCC 2038 TCC 2272 indica TCC 2360

3 3 indica TCC 2269 Ferlac indica TCC 2271 Ferlac salamae TCC 1996 salamae TCC 2217 salamae TCC 2270 Canastel salamae TCC 2037 Humber salamae TCC 2353 Tranoroa Salmonella Enteritidis TCC 1638 Salmonella Florida TCC 2288 Salmonella Gallinarum TCC 1641 Salmonella Give TCC 1642 Salmonella Hadar TCC 1644 Salmonella Heidelberg TCC 2011 Salmonella Hindmarsh TCC 1648 Salmonella Ibadan TCC 2297 Salmonella Indiana TCC 2295 Salmonella Infantis TCC 1650 Salmonella Inverness TCC 2281 Salmonella Java TCC 1652 Salmonella Javiana TCC 2291 Salmonella Kedougou TCC 2474 Salmonella Kentucky TCC 1653 Salmonella Kiel TCC 1654 Salmonella Kottbus TCC 2004 Salmonella Krefeld TCC 1655 Salmonella Lille TCC 2021 Salmonella Livingstone TCC 2005 Salmonella London TCC 1656 Salmonella Madelia TCC 2030 Salmonella Mbandaka TCC 2010 Salmonella Montevideo TCC 1657 Salmonella Moscow TCC 1658 Salmonella Muenchen TCC 1660 Salmonella Napoli TCC 2024 Salmonella Narashino TCC 1661 Salmonella Newport TCC 1663 Salmonella Niloese TCC 1664 Salmonella Nitra TCC 1693 Salmonella Ohio TCC 2000 Salmonella Oranienberg TCC 2015 Salmonella Orion TCC 2026 Salmonella Panama TCC 1665

4 4 Salmonella Paratyphi A NCTC 5702 Salmonella Paratyphi B OCC 149 Salmonella Pensacola TCC 2277 Salmonella Plymouth TCC 1994 Salmonella Pomona TCC 1988 Salmonella Poona TCC 1611 Salmonella Pretoria TCC 2029 Salmonella Pullorum TCC 1666 Salmonella Reading TCC 2006 Salmonella Rio-grande TCC 2285 Salmonella Rostock TCC 1667 Salmonella Rubislaw TCC 2017 Salmonella Saintpaul TCC 2007 Salmonella Senftenberg TCC 2018 Salmonella Shanghai TCC 2025 Salmonella Simsbury TCC 1672 Salmonella Stanley TCC 1673 Salmonella Taksony TCC 1678 Salmonella Tennessee TCC 2293 Salmonella Thompson TCC 2012 Salmonella Typhi NCTC 8385 Salmonella Typhimurium TCC 1681 Salmonella Umbilo TCC 1992 Salmonella Urbana TCC 1990 Salmonella Utrecht TCC 2298 Salmonella Vellore TCC 2290 Salmonella Virchow TCC 1890 Salmonella Wandsworth TCC 1685 Salmonella Westertede TCC 1686 Salmonella Zanzibar TCC 1690

5 5 Table 2: AOAC-RI exclusivity results for. Citrobacter freundii TCC 171 Negative Citrobacter intermedius TCC 181 Negative Citrobacter koseri TCC 2039 Negative Citrobacter youngae TCC 2043 Negative Edwardsiella tarda TCC 2216 Negative Enterobacter aerogenes TCC 2200 Negative Enterobacter amnigenus TCC 2198 Negative Enterobacter cloacae TCC 401 Negative Enterobacter faecalis TCC 1388 Negative Enterobacter intermedius TCC 2203 Negative Enterobacter sakazakii TCC 2053 Negative Escherichia blattae TCC 2273 Negative Escherichia coli TCC 1809 Negative Escherichia coli O157:H7 VT negative OCC 1872 Negative Escherichia fergusonii TCC 2263 Negative Escherichia hermanii TCC 2047 Negative Escherichia vulneris TCC 2264 Negative Hafnia alvei TCC 2044 Negative Klebsiella aerogenes TCC 1804 Negative Klebsiella oxytoca TCC 593 Negative Klebsiella pneumoniae TCC 1892 Negative Klebsiella terrigena TCC 2207 Negative Morganella morganii TCC 1431 Negative Pantoea agglomerans TCC 409 Negative Proteus mirabilis TCC 1566 Negative Proteus vulgaris TCC 1552 Negative Providencia alcalifaciens TCC 2209 Negative Providencia rettgeri TCC 2201 Negative Providencia stuartii TCC 418 Negative Pseudomonas aeruginosa TCC 1903 Negative Serratia liquifaciens TCC 2048 Negative Serratia marcescens TCC 414 Negative Shigella boydii TCC 2050 Negative Shigella flexneri TCC 2052 Negative Shigella sonnei TCC 2051 Negative Yersinia enterocolitica TCC 2215 Negative thermoscientific.com/suretect Conclusions The inclusivity and exclusivity study conducted as part of this AOAC-RI PTM validation study demonstrated that the SureTect Salmonella species PCR Assay showed 100% inclusivity and 100% exclusivity with all of the isolates analyzed Thermo Fisher Scientific Inc. All rights reserved. AOAC and AOAC Certification Mark are trademarks of and Performance Tested Methods is a service mark of AOAC International. NCTC is a trademark of Public Health England in the UK. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Contact Information: microbiology@thermofisher.com USA International +44 (0) LT2148A November 2014