PATHfinder. User Manual. PATHfinder Salmonella spp. Assay. Real-Time PCR kit for Salmonella spp. detection. Cat. # PMB01A/PMB01A-50

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1 PATHfinder PATHfinder Salmonella spp. Assay Real-Time PCR kit for Salmonella spp. detection Cat. # PMB01A/PMB01A-50 User Manual Via San Geminiano, San Prospero (MO)- Italy : : : laboratorio@generon.it : [1] User Manual - PATHfinder Salmonella spp. Assay Rev. 4-10/11/2016

2 1 - Introduction Salmonella is a genus of bacteria that are a major cause of foodborne illness throughout the world. It was isolated first in 1886 from americans doctor Daniel Elmer Salmon. Salmonella species present more than 2000 serotypes but the major worldspread serotypes as recorded from food analysis are S. enteritidis e S. typhimurium which are responsable of more than 50% of the foodborne diseases. S. enteritidis e S. typhimurium infection are caracherized by a typical gastroenterical evolution. These two serotypes are generally transmitted to humans through consumption of contaminated food of animal origin, mainly meat, poultry, eggs and milk. Gastroenteritis symptoms of Salmonella infection usually appear hours after infection, and include fever, abdominal pain, diarrhoea, nausea and sometimes vomiting. The illness usually lasts 4 7 days, and most people recover without treatment. However, in the very young and the elderly, and in cases when the bacteria enter the bloodstream, antibiotherapy may be needed before succumbing in serious issues. Some other Salmonella serotypes as S. typhi e S. paratyphi are involved in more severe illness as typhoid fever where, for these case, humans are the only host. The PATHfinder Salmonella spp. Detection Assay is a Real-Time PCR based technology platform analysis allowing identification of Salmonella isolates and detection (presence/absence) of Salmonella spp. in preenriched food samples. This assay is based on the peer reviewed article published by Gonzalez-Escalona et al. in Primer and probe sequences for the detection of Salmonella were designed on its specific inva gene. Generon in-house validation, the LOD for PATHfinder Salmonella spp. Detection Assay was experimentally determined to be between 1 and 10 copies. DNA was extracted using ION Liquid Force (Cat. # EXD008) and Generon ION Force DNA Extractor FAST (Cat. # EXD001). [2] User Manual - PATHfinder Salmonella spp. Assay Rev. 4-10/11/2016

3 2 - PATHfinder Salmonella spp. Assay This real-time PCR assay detects Salmonella spp. DNA. The amplification of the target sequences is measured by the use of a specific fluorescence-labeled probe (FAM) in less than 1.5 hours. An additional detection channel (HEX) detects the Internal Amplification Control (IAC) to exclude false negative results due to a PCR inhibition. When used along with PATHfinder Salmonella Amplicon DigiCount (Cat. # PMB01R) it is possible to quantify the genomic copies of Salmonella present in the unknown samples. The product is available in 50 or 100 reactions format Assay Content Box 50 reactions Box 100 reactions N. vials Volume (μl) N. vials Volume (μl) GENERase PLUS Mastermix PATHfinder OLIGO Mix * (OLIGOS and Probe pre-blended mix) Positive Control Negative Control * Reagents are supplied with a 5% of extra volume Storage & Expiry information Expiry date: see date on the packaging, product validity refers to the product kept intact in its original packaging. Protect reagents from light exposure as far as OLIGO Mix reagents are photosensitive. Store frozen. [3] User Manual - PATHfinder Salmonella spp. Assay Rev. 4-10/11/2016

4 3 Materials and equipment needed 3.1 Isolation and colony picking Material/Equipment Buffered Peptone Water (BPW) Sterilized Needles/Toothpicks Source 3.2 Extraction Material/Equipment Extraction Kit Vortexer Thermal Water Bath or Heat block for 1.5 ml tubes Pipette sets Pipette tips (Barrier) Tube rack for 1.5 ml tubes Source Generon GENERlex ION-Liquid Force 2.0 and 1.5 ml micro-tubes Micro centrifuge for ml micro-tubes 3.3 Detection via Real-Time PCR Material/Equipment Source Real-Time PCR System (1) PATHfinder Salmonella spp.assay Generon (PMB01A) Optical Adhesive Seal or Optical Caps Optical reaction plate or Optical Tube Strips Micropipette sets (1) The kit was validated on Bio-Rad CFX Deep-Well. The kit is compatible with other Real-time PCR instruments excluded Roche Light Cyclers I and II. [4] User Manual - PATHfinder Salmonella spp. Assay Rev. 4-10/11/2016

4 DNA extraction Each step of sample preparation (grinding, transferring, weighing, etc.) must be done according to GLP to minimize risks of cross-contamination between samples.

5 4 DNA extraction Each step of sample preparation (grinding, transferring, weighing, etc.) must be done according to GLP to minimize risks of cross-contamination between samples. It is recommended to use disposable tools whenever possible. The PATHfinder Salmonella spp. assay can be used for different purposes. First, in order to test food samples proceed weighing, into a proper microbiological enrichment bag, 25 g (or ml) of homogenized food sample in 225 ml of enrichment broth (e.g. Buffered Peptone Water BPW): incubate the bag at 37 C for 18 hours. After enrichment phase, operator can proceed with DNA extraction steps as described in the Quick Reference Guide for GENERlex ION-Liquid Force (Cat. # EXD008). To perform this technique no DNA extraction is needed. It is sufficient to pick one colony with a sterilized needle and to resuspend it in 1 X Reaction Mix before running the PCR. Colony PCR Diagram Pick a colony Resuspend in 1x working Mix Run PCR For the extraction of DNA from water and other samples using Bio-Rad Aquadien or Generon ION FORCE DNA Extractor FAST refer to these products user manual. [5] User Manual - PATHfinder Salmonella spp. Assay Rev. 4-10/11/2016

6 5 Real-Time PCR detection 5.1 Reaction setup I. Allow the reagents to thaw (GENERase PLUS Mastermix, PATHfinder OLIGO MIX, Positive Control and Negative Control). Vortex tubes when thawed and spin to collect contents at the bottom of the vial. II. III. IV. Mix 150 µl of PATHfinder OLIGO Mix with 750 µl of GENERase PLUS Mastermix to prepare PATHfinder Working Mastermix (WMX). Vortex briefly and spin down in order to homogenize the mix. Transfer 18 µl of WMX into each well. V. Add 12 µl of Negative Control into wells acting as negative controls. VI. VII. VIII. IX. When performing a Colony PCR experiment, add 12 µl of DNAse/RNAse Free Water for each well then pick one colony of interest with a sterilized needle from the agar plate and suspend it inside the selected well. Otherwise add 12 µl of the DNA extracted from the unknown samples using Bio-Rad Aquadien or Generon ION FORCE FAST DNA Extractor. Add 12 µl of Positive Control into wells acting as positive control. Seal plate/tubes with optical caps or film. Spin briefly optical PCR tubes or plates ensuring no bubbles are present at the bottom of the wells. 5.2 Instrument setup Set the following parameters on your thermocycler: I. Total Reaction volume: 30 µl II. III. Fluorophores/Quenchers: Salmonella spp. (FAM/BHQ1-NFQ); Internal Amplification Control (HEX/BHQ1-NFQ). Use VIC/JOE calibrations in case the Real Time Instrument does not possess a HEX calibrated reading channel. Thermal profile: Step T ( C) Duration Loops UNG 50 2 min 1 Taq Activation min 1 Denaturation sec Annealing/Extension + Plate Reading sec 45 [6] User Manual - PATHfinder Salmonella spp. Assay Rev. 4-10/11/2016

7 6 Data interpretation After performing PCR, each individual sample is analyzed through the instrument software to produce a Cq value (quantification cycle) for each reporter dye. These values are used to determine the presence (Qualitative Test) of pathogen into the sample. See below an example of the graphics obtained for a positive Salmonella spp. and for a non-salmonella spp. (Fig. B) After setting the baseline, the analysis outcome should be evaluated following the indications below. If the following conditions are met: TEST Salmonella spp. (FAM) Internal Amplification Control (HEX) Positive Control + Not significative Negative Control - + Then the possible results for any sample are: TEST Salmonella spp. (FAM) Internal Amplification Control (HEX) Positive Salmonella spp. + Not significative Negative Salmonella spp. - + Inhibited sample - - In case of inhibition refer to the troubleshooting section of this guide before performing a new test. [7] User Manual - PATHfinder Salmonella spp. Assay Rev. 4-10/11/2016

8 7 Inclusivity and Exclusivity Panel The following DNA extracts showed amplification curves in FAM channel: S. enterica subsp. enterica serovar Agona, S. enterica subsp. enterica serovar Heidelberg, S. enterica subsp. enterica serovar Muenchen, S. enterica subsp. enterica serovar Braenderup, S. enterica subsp. enterica serovar Kentucky, S. enterica subsp. enterica serovar Newport, S. enterica subsp. enterica serovar Brandenburg, S. enterica subsp. enterica serovar Livingstone, S. enterica subsp. enterica serovar Ohio, S. enterica subsp. enterica serovar Bredeney, S. enterica subsp. enterica serovar London, S. enterica subsp. enterica serovar Saintpaul, S. enterica subsp. enterica serovar Derby, S. enterica subsp. enterica serovar Mbandaka, S. enterica subsp. enterica serovar Schwarzengrund, S. enterica subsp. enterica serovar Give, S. enterica subsp. enterica serovar Montevideo, S. enterica subsp. enterica serovar Johannesburg, S. enterica subsp. enterica serovar Abortusequi,S. enterica subsp. enterica serovar Durban, S. enterica subsp. enterica serovar Kotte, S. enterica subsp. enterica serovar Alamo, S. enterica subsp. enterica serovar Enteritidis, S. enterica subsp. enterica serovar Lexington, S. enterica subsp. enterica serovar Albany,S. enterica subsp. enterica serovar Eastbourne, S. enterica subsp. enterica serovar Lille, S. enterica subsp. enterica serovar Alachua, S. enterica subsp. enterica serovar Edinburg, S. enterica subsp. enterica serovar Limete, S. enterica subsp. enterica serovar Brisbane, S. enterica subsp. enterica serovar Fresno, S. enterica subsp. enterica serovar Lindenburg, S. enterica subsp. enterica serovar Amsterdam, S. enterica subsp. enterica serovar Friedenau, S. enterica subsp. enterica serovar Luciana, S. enterica subsp. enterica serovar Augustenborg, S. enterica subsp. enterica serovar Gaminara, S. enterica subsp. enterica serovar Meleagridis, S. enterica subsp. enterica serovar Brunei, S. enterica subsp. enterica serovar Gatuni, S. enterica subsp. enterica serovar Madelia, S. enterica subsp. enterica serovar California, S. enterica subsp. enterica serovar Gera, S. enterica subsp. enterica serovar Matopeni, S. enterica subsp. enterica serovar Carrai, S. enterica subsp. enterica serovar Hadar, S. enterica subsp. enterica serovar Muenster, S. enterica subsp. enterica serovar Cerro, S. enterica subsp. enterica serovar Hartford, S. enterica subsp. enterica serovar Mgulani, S. enterica subsp. enterica serovar Charity, S. enterica subsp. enterica serovar Havana, S. enterica subsp. enterica serovar Michigan, S. enterica subsp. enterica serovar Chester, S. enterica subsp. enterica serovar Hildago, S. enterica subsp. enterica serovar Minnesota, S. enterica subsp. enterica serovar Chingola, S. enterica subsp. enterica serovar Hilversum,S. enterica subsp. enterica serovar Mississippi, S. enterica subsp. enterica serovar Cubana, S. enterica subsp. enterica serovar Hvittingfoss, S. enterica subsp. enterica serovar Manhattan, S. enterica subsp. enterica serovar Daytona, S. enterica subsp. enterica serovar Infantis, S. enterica subsp. enterica serovar Molade, S. enterica subsp. enterica serovar Bangkok, S. enterica subsp. enterica serovar Inverness,S. enterica subsp. Salamae serovar Mobeni, S. enterica subsp. enterica serovar Bareilly, S. enterica subsp. enterica serovar Isangi, S. enterica subsp. Salamae serovar Mjimwema, S. enterica subsp. enterica serovar Bournemouth, S. enterica subsp. enterica serovar Javiana, S. enterica subsp. Salamae serovar Negev, S. enterica subsp. enterica serovar Nagoya, S. enterica subsp. enterica serovar Poona, S. enterica subsp. arizonae serovar Arizonae, S. enterica subsp. enterica serovar Nchanga, S. enterica subsp. enterica serovar Ramatgan, S. enterica subsp. Diarizonae serovar Diarizonae, S. enterica subsp. enterica serovar Norwich, S. enterica subsp. enterica serovar Reading, S. enterica subsp. houtenae serovar Sachsenwald, S. enterica subsp. enterica serovar Nottingham, S. enterica subsp. enterica serovar Rubislaw, S. bongori serovar Brookfield, S. enterica subsp. enterica serovar Oranienburg, S. enterica subsp. enterica serovar Ruiru,S. enterica subsp. enterica serovar Orion,S. enterica subsp. enterica serovar Saphra, S. enterica subsp. enterica serovar Paratyphi B, S. enterica subsp. enterica serovar Stanley, S. enterica subsp. enterica serovar Putten, S. enterica subsp. enterica serovar Typhi. The following DNA extracts showed amplification curve only in HEX channel: Alicyclobacillus acidiphilus (DSMZ 14558), Alicyclobacillus acidocaldarius subsp. Acidocaldarius (ATCC 27009), Alicyclobacillus acidoterrestris (ATCC 49025),Bacillus cereus (ATCC 14579), Bacillus cereus cereulide (DSMZ 4312), Bacillus subtilis subsp. spitzenii(atcc 6633), Campylobacter coli (ATCC 33559), Campylobacter fetus (ATCC 27374), Campylobacter jejuni (ATCC 33560), Campylobacter laridis (ATCC 35221), Citrobacter freundii (ATCC 8090), Clostridium perfringens (ATCC 25768), Clostridium sporogenes (ATCC 3584), Cronobacter sakazakii (ATCC 29544), Edwardsiella tarda (ATCC 15947), Proteus mirabilis (ATCC 29906), Citrobacter koseri (ATCC 27028)Aeromonas hydrophila, Escherichia coli (ATCC 11775), Janthinobacterium lividum (ATCC 12473), Klebsiella pneumoniae (ATCC 13883), Lactococcus lactis subsp. lactis (ATCC 19435), Legionella pneumophila serovar 1 (ATCC 33152), Listeria innocua (ATCC 33090), Listeria ivanovii (ATCC 19119), Listeria monocytogenes (ATCC 15313), Morganella morganii subsp. morganii (ATCC 25830), Proteus vulgaris (ATCC 29905), Providencia stuartii (ATCC 29914), Pseudomonas aeruginosa (ATCC 10145), Pseudomonas fluorescens (ATCC 13525), Pseudomonas syringae (ATCC 19310), Proteus vulgaris (ATCC 13315), Pseudomonas fragii (ATCC 4973), Enterobacter cloacae (ATCC 13047), Serratia marcescens (ATCC 13880), Shigella boydii (ATCC 8700), Shigella flexneri (ATCC 29903), Shigella sonnei (ATCC 29930), Staphylococcus aureus (ATCC 12600), Vibrio aeregenes (ATCC ), Vibrio algynoliticus (ATCC 17749), Vibrio mytili (ATCC 51288), Vibrio parahaemolyticus (ATCC 17802), Vibrio splendidus (ATCC 33125), Vibrio vulnificus (ATCC 27560), Yersinia enterocolitica subsp. Enterocolitica (ATCC 23715), Yersinia pseudotuberculosis (ATCC 29833). [8] User Manual - PATHfinder Salmonella spp. Assay Rev. 4-10/11/2016

9 8 Troubleshooting I. Concomitant no target nor IAC amplification, or amplification plots grossly abnormal. Possible causes and corrective actions: An excess of DNA and/or PCR inhibitors inhibits the reaction I. Pick less bacteria sludge or dilute it 1:10 after colony picking. II. Dilute 1:10 the DNA extracted using Bio-Rad Aquadien or Generon ION FORCE FAST DNA Extractor Inadequate sealing of optical caps/film caused sample evaporation. Redo the analysis using proper tools and proper optical caps/film to secure perfect sealing. Inadequate consumables. Redo the analysis and use only optical grade 96-well plates and optical adhesive seal or optical 8-well strips and caps. II. Positive Control reactions failed to amplify, but other reactions appear correct (e.g. the IAC is amplified): Positive Control DNA was not added to the reaction wells. If other reactions look normal, there may be no need to repeat the run. III. Negative Control reactions are positive: Contamination of the negative control vial or the PATHfinder PCR mix with PATHfinder positive DNA. Use more care to prevent contamination while handling assay reagents and setting up assays. In case support is needed contact Generon at: 9 Disclaimers Generon S.p.A. guarantees the buyer exclusively concerning the quality of reagents and of the components used to manufacture the product. Generon S.p.A. is not responsible and cannot anyway be considered responsible or jointly responsible for any possible damages resulting from the utilization of the product by the user and from the data obtained. [9] User Manual - PATHfinder Salmonella spp. Assay Rev. 4-10/11/2016

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