foodproof Saccharomyces cerevisiae var. diastaticus Detection Kit - Hybridization Probes (LC 1.x, 2.0) -

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1 For food testing purposes FOR IN VITRO USE ONLY foodproof Saccharomyces cerevisiae var. diastaticus Detection Kit - Hybridization Probes (LC 1.x, 2.0) - Version 3, September 2017 PCR kit for the qualitative detection of Saccharomyces cerevisiae var. diastaticus DNA using the LightCycler Carousel Based System Order No. R Kit for 96 reactions for a maximum of 90 samples. Store the kit at -15 to -25 C

2 Table of Contents 1. Storage and Stability Kit Content Use of the Internal Amplification Control Additional Equipment and Reagents Required 4 5. Standard Protocol Program Setup Interpretation (Amplification) Interpretation (Melting Curve) License Remarks Trademark Change Index 9 2

3 This kit is designed for the rapid detection and identification of the obligatory beer spoilage yeast Saccharomyces cerevisiae var. diastaticus. Recommended for sample preparation is the foodproof StarPrep Four Kit (S ). 1. Storage and Stability The kit is shipped on dry ice. Store the kit at 15 C to 25 C through the expiration date printed on the label. Once the kit is opened, store the kit components as described in the following Kit Contents table: 2. Kit Content Every detection system contains ready to use chemicals for 96 reactions with a reaction volume of 20 µl. Vial/Cap Color Label Contents/Function/Storage 1 yellow cap foodproof Saccharomyces cerevisiae var. diastaticus Master Mix 2 red cap foodproof Saccharomyces cerevisiae var. diastaticus Enzyme Solution 3 white cap foodproof Saccharomyces cerevisiae var. diastaticus Internal Control 4 purple cap foodproof Saccharomyces cerevisiae var. diastaticus Control Template 3 x 420 µl Ready-to-use primerand HybridizationProbes mix specific for S. c. var. diastaticus DNA and the S. c. var. diastaticus Internal Control and PositiveControl. For amplification and detection of S. c. var. diastaticus specific sequences. NOTE: The foodproof Saccharomyces cerevisiae var. diastaticus Master Mix contains fluorescence labeled probes; therefore do not extensively expose them to light! Do not freeze and thaw reagents more than twice! Store at -15 to -25 C. Shelf life at this temperature is 12 months. 3 x 32 µl Contains Fast Start Taq DNA Polymerase and Uracil-DNA Glycosylase (heat labile) for preventionof carry-overcontamination. Store at -15 to -25 C. Shelf life at this temperature is 12 months. 3 x 32 µl Contains a stabilized solution of plasmid DNA. For use as an internal amplification control. Store at -15 to -25 C. Shelf life at this temperature is 12 months. The Internal Control can be stored at 2 to 8 C for up to 1 month after thawing. 1 x 50 µl Contains a stabilized solution of plasmid DNA. For use as a PCR run positivecontrol. Store at -15 to -25 C. Shelf life at this temperature is 12 months. The Control Templatecan be stored at 2 to 8 C for up to 1 month after thawing. 5 colorless cap H 2 O, PCR-grade 1 x 1 ml Nuclease-free, PCR-grade H 2 O. For use as a PCR-run negative control (no template control). Store at -15 to -25 C. Shelf life at this temperature is 12 months. 3

4 3. Use of the Internal Amplification Control To prevent false negative results e.g. due to PCR inhibition, the amplification is carried out as a competitive PCR by addition of the Internal Control. In case of a false negative result due to PCR inhibition by DNA elute the amplification of the Internal Control also is suppressed. The undisturbed amplification of the Internal Control proves that the negative result is due to the absence of DNA from the beer spoilage yeast Saccharomyces cerevisiae var. diastaticus. 4. Additional Equipment and Reagents Required To perform assays with the foodproof Saccharomyces cerevisiae var. diastaticus Detection Kit, Hybridization Probes (LC 1.x, 2.0), you will need the following equipment. LightCycler 1.x Instrument or LightCycler 2.0 Instrument (Roche Diagnostics: Cat. No or Cat. No ) 2 LightCycler Capillaries (Roche Diagnostics: Cat. No ) 2 LightCycler - Color Compensation Set (Roche Diagnostics: Cat. No ) 2 Nuclease-free aerosol-resistant pipette tips Pipettes with disposable, positive-displacement tips Sterile reaction (Eppendorf) tubes for preparing master mixes and dilutions Standard benchtop microcentrifuge containing a rotor for 2.0 ml reaction tubes foodproof StarPrep Four Kit 1 To ease the spinning of the reaction mixes into the LightCycler Capillaries, centrifuge the LightCycler Sample Carousel (loaded with the filled capillaries) in the LC Carousel Centrifuge (Roche: Cat. No ). 2 1 Available from BIOTECON Diagnostics 2 Available from Roche Diagnostics Note: Centrifuge adapters, which can be used in a standard microcentrifuge rotor, are supplied with the LightCycler Instrument. 5. Standard Protocol It is recommended to use filter tips! 1. Determine the total number of PCR reactions (sample and control reactions) and calculate the required volumes. In addition to the number of samples, two control reactions (negative control and positive control) should be included in each LightCycler run. Due to pipetting errors it is recommended to prepare the volumes of Master Mix, Enzyme Solution and Internal Control for 1 2 reactions additionally. An example is given in Table 1. 4

5 Table 1: Example for the preparation of the reactions Note: The volumes indicated below are based on a single 20 µl standard reaction. Prepare the PCR mix by multiplying the amount in the Volume column by the number of reactions to be cycled plus one or two additional reactions. Component Volume foodproof Saccharomyces cerevisiae var. diastaticus Master Mix, (vial 1, yellow cap) 13 µl foodproof Saccharomyces cerevisiae var. diastaticus Enzyme Solution (vial 2, red cap) 1.0 µl foodproof Saccharomyces cerevisiae var. diastaticus Internal Control (vial 3, white cap) 1.0 µl Total volume 15.0 µl 2. Depending on the total number of reactions, place LightCycler Capillaries in the centrifuge adapters. 3. Thaw, mix gently (do not vortex!) but thoroughly and centrifuge all reagents. 4. Transfer the calculated volume of Master Mix into a new sterile reaction tube. Add the corresponding volume of Enzyme Solution and Internal Control to the Master Mix. Mix thoroughly by pipetting. 5. Transfer 15.0 µl of PCR mix including Internal Control into all prepared capillaries. 6. For the samples of interest, add up to 5 µl sample DNA (if less than 5 µl add H 2 O to 5 µl) to a capillary. Seal the capillaries with a stopper. 7. For the negative control, add 5 µl PCR-grade H 2 O (vial 4, colorless cap). Seal the capillary with a stopper. 8. For the positive control, add 5 µl foodproof Saccharomyces cerevisiae var. diastaticus Control Template (vial 4, purple cap). Seal the capillary with a stopper. 9. Place capillaries into adapters and centrifuge at 700 x g for 5 seconds (3,000 rpm in a standard bench-top microcentrifuge). 10. Place the capillaries in the rotor of the LightCycler -Instrument. 11. Start the PCR run. 5

6 6. Program Setup The amplification and Melting Curve generation with the LightCycler -1.x Instrument or LightCycler 2.0 Instrument is carried out according to the following temperature-time-program. Pre-incubation Programs/Cycle Program Data Value Cycles 1 Analysis Mode None Temperature Targets Segment 1 Segment 2 Target/Target Temperature [ C] Hold/Incubation Time [h:min:s] 00:02:00 00:15:00 Ramp Rate/Temperature Transition Rate [ C/s] Sec Target/Secondary Target Temperature [ C] 0 0 Step Size [ C] Step Delay [cycles] 0 0 Acquisition Mode None None 6

7 Amplification Programs/Cycle Program Data Value Cycles 45 Analysis Mode Quantification Temperature Targets Segment 1 Segment 2 Segment 3 Target/Target Temperature [ C] Hold/Incubation Time [h:min:s] 00:00:02 00:00:30 00:00:15 Ramp Rate/Temperature Transition Rate [ C/s] Sec Target/Secondary Target Temperature [ C] Step Size [ C] Step Delay [cycles] Acquisition Mode None Single None Melting Curve Analysis Programs/Cycle Program Data Value Cycles 1 Analysis Mode Melting Curves Temperature Targets Segment 1 Segment 2 Segment 3 Target/Target Temperature [ C] Hold/Incubation Time [h:min:s] 00:00:00 00:01:00 00:00:00 Ramp Rate/Temperature Transition Rate [ C/s] Sec Target/Secondary Target Temperature [ C] Step Size [ C] Step Delay [cycles] Acquisition Mode None None Cont 7

8 Cooling Programs/Cycle Program Data Value Cycles 1 Analysis Mode Melting Curves Temperature Targets Segment 1 Target/Target Temperature [ C] 40 Hold/Incubation Time [h:min:s] 00:00:30 Ramp Rate/Temperature Transition Rate [ C/s] 20 Sec Target/Secondary Target Temperature [ C] 0 Step Size [ C] 0.0 Step Delay [cycles] 0 Acquisition Mode None 7. Interpretation (Amplification) The specific amplification of DNA of Saccharomyces cerevisiae var. diastaticus and the amplification of the foodproof Saccharomyces cerevisiae var. diastaticus Control Template is analyzed in fluorescence channel F2/Back- F1(LightCycler 1.x Instrument) or 640/Back 530 (LightCycler 2.0 Instrument). The specific amplification of the Internal Control is analyzed in fluorescence channel F3/Back-F1 (LightCycler 1.x Instrument) or 705/Back 530 (LightCycler 2.0 Instrument). 8. Interpretation (Melting Curve) A Melting Curve Analysis is performed in case of a positive result in order to differentiate the melting peaks of sample DNA and the Control Template. The melting peak pattern of the Control Template and sample DNA is clearly different. The melting peaks temperatures are not identical. The Control Template has a main peak at approx. 58 C and a shoulder at approx. 62 C. The melting peaks of Saccharomyces cerevisiae var. diastaticus positive samples are at approx. 62 C (main Peak) and approx. 67 C. The peak height of Saccharomyces cerevisiae var. diastaticus positive samples may vary according to the initial DNA concentration. If the initial DNA concentration is high there might be only one peak. All Saccharomyces cerevisiae var. diastaticus positive samples have a clear peak area above 66 C and no peak at 58 C independent of the initial DNA concentration. A guarantee for the differentiation by melting curves cannot be given. 8

9 9. License Notice The purchase price of this product includes limited, nontransferable rights under U.S. Patent No. 7,687,247 owned by Life Technologies Corporation to use only this amount of the product to practice the claims in said patent solely for activities of the purchaser for bioburden testing, environmental testing, food testing, or testing for genetically modified organisms (GMO) in accordance with the instructions for use accompanying this product. No other rights are conveyed, including no right to use this product for in vitro diagnostic, therapeutic, or prophylactic purposes. Further information on purchasing licenses under the above patent may be obtained by contacting the Licensing Department, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA Trademarks foodproof is a trademark of BIOTECON Diagnostics GmbH. LIGHTCYCLER and HYBPROBE are trademarks of Roche. Other brand or product names are trademarks of their respective holders. 11. Change Index Version 1, August 2008: First version of the package insert. Version 2, March 2017: License Notice changed. Version 3, September 2017: License Notice changed. 9

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12 R (10) BIOTECON Diagnostics GmbH Hermannswerder Potsdam Germany Phone +49 (0) Fax +49 (0)

foodproof Escherichia coli and Shigella Detection Kit - Hybridization Probes (LC 1.x, 2.0, 480 II) -

For food testing purposes. FOR IN VITRO USE ONLY. foodproof Escherichia coli and Shigella Detection Kit - Hybridization Probes (LC 1.x, 2.0, 480 II) - Version 3, September 2017 PCR kit for the qualitative