4e For everyone Detection Kit Y Dekkera (Brettanomyces) sp. Screening

Transcription

1 4e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening USER GUIDE Detectin f all Dekkera (Brettanmyces) Yeasts Fr research use nly Catalg # # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 1 f 12

2 Table f Cntents Sectin e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening... 3 Sectin Intrductin t the 4e Fr everyne Detectin Kit Technlgy... 3 Sectin Kit Cmpnents... 4 Sectin Shelf Life and Strage... 4 Sectin Materials Required but Nt Supplied... 4 Sectin Overview... 5 Sectin Detailed Instructins... 6 Sample Preparatin... 6 DNA Analysis by real time PCR... 7 Preparatin and Distributin f the Recnstitutin Slutin... 7 Instrument and Sftware Setup... 8 Data Analysis... 8 Sectin Precautins and Recmmendatins fr Best Results... 9 Sectin 9 Appendix PCR Mix Calculatin Guide Trademarks and Prperty Rights # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 2 f 12

3 Sectin 1 4e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening This test allws the quantitative detectin f all species f Dekkera (Brettanmyces) in ne test. The species range includes D. anmala, D. bruxellensis, D. custersianus, D. naardenensis and D. nanus. Brettanmyces belngs t the super attenuating yeasts and can grw in finished beer, prducing carbn dixide. It is used in sur beer prductin, but is regarded as a spiler in cnventinal beers. It ften takes mnths until spilage suddenly ccurs in finished prducts. The taste f cntaminated beer is sur and ften unpleasant, reminding f hrse sweat, and the pressure can grw up t causing explsin f packaged units. All Brettanmyces species are detected tgether in ne screening test which prvides a simple and easy result read-ut fr the presence f Brett in yur sample. Sectin 2 Intrductin t the 4e Fr everyne Detectin Kit Technlgy Tday, the use f PCR is accepted as the standard methd fr detecting nucleic acids frm numerus micrrganisms in a diversity f fd and beverages, bth functinal species as well as spilers. Real time PCR is ne f the mst pwerful, specific and reliable methds fr the quantitative detectin and identificatin f micrrganisms at an early prcess stage t prevent spilage and t maintain verall prduct quality. The 4e Fr everyne Detectin Kit system is based n DNA amplificatin and detectin f micrrganisms by real time PCR. The specific PCR reagents, primers and prbes, cme in a ready-made dried frmat in the PCR tubes fr unrivalled ease f use and temperature stability. All PCR tests use the FAM channel (494/518 nm) fr detectin f the target micrrganisms and the HEX channel (535/556 nm) fr an internal cntrl reactin. This allws 4e Fr everyne Detectin Kits t prevent false negatives due t sample inhibitin, allwing yu t be truly cnfident abut negative results. A typical wrkflw includes the fllwing fur steps: Enrichment (ptinal) DNA Extractin Real time PCR Data Analysis & Interpretin Data analysis is ptimized fr use with the Chai Dual Channel Open qpcr real time PCR thermcycler ( In rder t achieve lwest detectin limits, we recmmend sample enrichment in ur PCR certified FastOrange enrichment media ( # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 3 f 12

4 Sectin 3 Kit Cmpnents The 4e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening kit cntains sufficient reagents fr 48 reactins. Kit material fr DNA islatin and analysis Amunt Strage Washing buffer A (yellw cap) Lysis buffer B (blue cap) Rehydratin buffer B (white cap) 2 x 10.0 ml 1 x 10.0 ml 1 x 5.0 ml Psitive cntrl DNA (red cap) 1 x 50 µl PCR tubes (strips f 8) with BHQmix 6 2 x Master mix (green cap) 1 x 850 µl 2-8 C Cap strips (strips f 8) fr cvering the PCR reactin tubes C r ambient Table 1: Materials supplied Sectin 4 Shelf Life and Strage Once received, the kit must be stred at 2-8 C. Reagents stred at this temperature can be used until the expiratin date indicated n the package label. Sectin 5 Materials Required but Nt Supplied Equipment Real time PCR thermcycler fr 0.1 ml tubes with detectin channels fr FAM (520 nm emissin) and HEX (550 nm emissin) Benchtp micrcentrifuge fr 1.5 ml reactin tubes, 10,000 rpm (RCF: 10,000 x g) minimum Centrifuge fr 8-tube strips 0.1 ml r adaptr fr benchtp micrcentrifuge Reactin tube mixer (Vrtexer) (ptinal) Thermincubatr, dry bath r water bath set t 80 C ± 5 C Micrliter pipettes fr DNA extractin 100-1,000 µl variable vlume 200 µl fixed vlume (ptinal) Micrliter pipettes fr PCR set-up 100-1,000 µl variable vlume 25 µl fixed vlume 5 µl fixed vlume, r variable equivalent Table 2: Additinal materials required Supplies 1.5 ml reactin tubes, 2 per sample plus 1 per run Pipette tips with filters Glves, pwder free Ideally use different pipettes fr DNA extractin and PCR set-up # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 4 f 12

5 Sectin 6 Overview Refer t Sectin 9 PCR Mix Calculatin Guide fr preparatin f recnstitutin slutin. # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 5 f 12

6 Sectin 7 Detailed Instructins Warning! Read the manual and the Safety Data Sheets befre starting the analysis. Safety Data Sheets are available in the dwnlad area frm All handling steps shuld be perfrmed under sterile cnditins. Wear apprpriate prtective clthing and pwder free glves. The use f filter tips is recmmended. Sample Preparatin Befre yu start: Heat water bath, dry bath r thermincubatr t 80 C 1. Transfer the samples int a 1.5 ml reactin tube: a) Liquid samples: - Clear samples (rinse water, filtered beer, enrichment withut visible grwth, etc): Standard vlume, sample after enrichment: Use ml. Prceed t step 2. Larger vlume, sample withut enrichment: use larger vlume, up t 50 ml: Centrifuge sample 5 min at 4,000-5,000 rpm (RCF: 2,700-3,000 x g) Slwly decant the liquid, leave abut 1 ml in the cne f the tube Mix liquid in the cne with a pipet tip and transfer it cmpletely int a 1.5 ml reactin tube Prceed t step 2. - Turbid samples, if turbidity caused by bacterial grwth (previusly enriched sample, yellw clr in FastOrange B, r cntaminated prduct): Use 50 µl. Prceed t step 2. - Turbid sample, if a high yeast cncentratin is knwn in the sample (fermenter, pitching yeast, unfiltered beer): Use µl t reach a pellet size f app. 2 mm in diameter after centrifugatin (see fig. 1). Prceed t step 2. b) Clnies: Single clnies as well as a cuple f different clnies tgether can be prcessed as ne sample First transfer 200 µl Washing buffer A int a 1.5 ml reactin tube Add cell material frm all clnies t be analyzed int the liquid. Prceed t step Centrifuge in a mini centrifuge fr 3 min at >10,000 rpm (RCF: 8,500 x g) r alternatively in a larger centrifuge fr 10 min at 4,000 5,000 rpm (RCF: 2,700 3,000 x g) 3. Cntrl the pellet sizes, which is the cell material, f yur samples. The pellet size shuld nt exceed 2 mm in diameter (see fig. 1). If necessary, remve part f the pellet tgether with the liquid phase in step Remve the liquid phase carefully and discard Fig. 1: recmmended pellet sizes Left: maximum pellet size fr turbidity frm bacteria Right: maximum pellet size fr yeast cntaining samples # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 6 f 12

7 5. Wash the pellet as fllws: Add 200 µl Washing buffer A t the pellet Resuspend pellet by vrtexing r pipetting up and dwn, and repeat steps Extended washing might be necessary fr samples knwn t likely be inhibited: Repeat the whle washing prcedure as abve, and/r Use a higher vlume f washing buffer - up t 1,000 µl per wash 6. Add 200 µl f Lysis buffer B and again resuspend the cell pellet thrughly 7. Incubate samples at 80 C ± 5 C fr 10 min in a thermincubatr, dry bath r water bath 8. Centrifuge again as in step 2. Attentin! D NOT discard supernatant nw as it cntains yur sample DNA! The pellet cntains cell debris and ther waste particles, which were separated frm the DNA 9. Use the liquid phase fr PCR, take care NOT t tuch the pellet in the bttm f the tube when pipetting 10. Fr vernight r lng term strage, transfer 100 µl f the liquid phase frm 8. int a new 1.5 ml reactin tube 11. Stre at 2-8 C fr same day PCR analysis; fr lng-term strage, freeze at -18 t -20 C DNA Analysis by real time PCR All reactin cmpnents fr PCR except the 2-fld cncentrated Master mix are prvided in a dried frm in the PCR tubes. Each tube cntains BHQmix which delivers primers and prbes fr the detectin f the target species plus the reagents fr the internal psitive cntrl (IPC). The number f PCR tubes and cap strips can be adjusted accrding t the number f samples t be analyzed by cutting the needed amunt with sterilized (flamed) scissrs r a knife. Remember t always add 2 tubes and caps fr the psitive and negative cntrls. Preparatin and Distributin f the Recnstitutin Slutin 1. Prepare ne PCR reactin tube fr each sample. Additinally ne psitive and ne negative cntrl reactin are always neccessary per run. Take care NOT t tuch caps r tubes when nt wearing glves! 2. Prepare the recnstitutin slutin by pipetting the required amunts f Rehydratin buffer and 2 x Master mix int a fresh 1.5 ml reactin tube. Refer t table 1 fr the vlumes. When using table 1, cnsider nly the number f samples yu want t analyze. D NOT cunt the cntrl reactins in yur calculatin The vlume needed fr the 2 cntrl reactins per run are already accunted fr in the table A 10% verage t accunt fr pipetting lsses is als included in the ttal calculatin 3. After adding bth slutins, clse the reactin tube, which nw cntains the recnstitutin slutin. Mix briefly by vrtexing r inverting the tube a cuple times. Fllw up with a quick spin dwn t cllect the liquid at the bttm f the tube 4. Pipet 25 µl f the recnstitutin slutin int each PCR tube needed (number f samples + 2 cntrls) # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 7 f 12

8 5. Prepare the cntrl reactins: Psitive cntrl: Pipet 5.0 µl f the prvided DNA int the first PCR tube. D nt add any sample. Negative cntrl: Pipet 5.0 µl Rehydratin buffer int the secnd PCR tube. D nt add any sample. 6. Prepare the samples: Pipet 5.0 µl f the extracted sample DNA (frm part: Sample Preparatin) int ne PCR tube each, starting with tube 3 7. Clse all PCR tubes with the prvided cap strips 8. Spin dwn the PCR tubes shrtly (10-15 secnds) t cllect the liquid at the bttm (max. 2,000 rpm), and check fr trapped air bubbles 9. If trapped bubbles are present, repeat step Transfer all PCR tubes int the thermcycler and fllw instructins accrding t the sftware Instrument and Sftware Setup Fr instrument and sftware setup, fllw instructins in the Open qpcr instrument sftware. If yu are using a different thermcycler instrument, set the fllwing prfile: step 1 step 2 95 C 95 C 2:00 0:15 60 C 1:00 Detectrs: FAM (520 nm emissin) HEX (550 nm emissin) Quencher: BHQ Data cllectin Sample vlume 30 µl All times given are in minutes:secnds Fig. 2: Temperature scheme f thermcycler Data Analysis 40x 1. Chai Dual Channel Open qpcr real time PCR thermcycler All data can be analyzed directly at the end f the PCR run r at a later time by pening the stred experiment. Fllw instructins in the crrespnding Open qpcr sftware manual fr pening experiments and setting the data analysis parameters Interpreting Results i. Once the run is cmplete, the sftware displays the full data interpretin in a table frmat which is the standard read-ut ii. The full data including amplificatin curves can be seen under the Full Results view 2. Other real time PCR thermcyclers Fllw user manual f thermcycler instrument Evaluate the thermcycler results as fllws: i. Verify the curves ii. Evaluatin f the measured Cq/Ct values: # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 8 f 12

9 Detectin f target (FAM channel) FAM channel detects target rganisms HEX channel detects internal psitive cntrl reactin Internal cntrl reactin (HEX channel) Result frm analysis + + Psitive: DNA f target (ref. Sectin 1) is present + - Psitive: DNA f target (ref. Sectin 1) is present - + Negative: DNA f target (ref. Sectin 1) is nt detected - - Table 3: Manually evaluating PCR results Result is nt evaluable: Either: Dilute extracted DNA with rehydratin buffer 1:1,000 and repeat PCR Or: Repeat the DNA extractin with a smaller amunt f sample, applying mre extensive washing refer t Sectin 8 Sectin 8 Precautins and Recmmendatins fr Best Results This test must be perfrmed by trained persns All ptentially infectius material shuld be autclaved befre dispsal The quality f results depends n strict cmpliance with Gd Labratry Practices (fr example, the EN ISO 7218 standard), especially cncerning PCR: The labratry equipment (pipets, tubes, etc.) must nt circulate frm ne wrkstatin t anther It is essential t use psitive and negative cntrls fr each series f amplificatin reactins D nt use reagents after their expiratin date Peridically verify the accuracy and precisin f pipets and the crrect functining f the instruments Change glves ften, especially if yu suspect they are cntaminated Clean wrk spaces peridically with at least 5% bleach r ther DNA decntaminating agents such as DNA AWAY Use pwder-free glves and avid fingerprints and writing n tube caps as this can interfere with data acquisitin It is strngly advised t fllw the general requirements described in the standard EN ISO 22174:2005 (Micrbilgy f fd and animal feeding stuffs Plymerase chain reactin (PCR) fr the detectin f fd pathgens General requirements and definitins) # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 9 f 12

10 Sectin 9 Appendix PCR Mix Calculatin Guide When using the Chai Dual Channel Open qpcr real time PCR thermcycler: T find the crrect vlumes t pipet when preparing the recnstitutin slutin, add the ttal number f samples t be analyzed, and find the crrespnding vlumes f Rehydratin buffer and 2 x Master mix in the table. Kit 1 vlume t pipet in µl ttal number f samples Rehydratin buffer 2x Master mix Vlume in µl Table 4: 1 Kit per Run Kit 1 + Kit 2 vlume t pipet in µl ttal sum f samples frm bth kits Rehydratin buffer 2x Master mix Vlume in µl Table 5: 2 Kits per Run # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 10 f 12

11 Trademarks and Prperty Rights Trademarks: 4e Fr everyne Detectin Kit, FastOrange and PIKA Weihenstephan are registered trademarks r trademarks f PIKA Weihenstephan, Pfaffenhfen, Germany, in Germany and ther cuntries. Chai and Open qpcr are trademarks f Chai Bitechnlgies Inc., Santa Clara, CA, USA. Use f prduct: 4e Detectin Kit is t be used fr research purpses nly. Prperty Rights: Fr any cmmercial use f the kit r parts f it, licensing frm PIKA Weihenstephan GmbH is required. The use f ur prducts may tuch prperty rights f third parties. The purchase f this prduct des nt implement any rights fr the perfrmance f PCR r its use fr diagnstic purpses. We pint ut that licensed accessries (thermcycler instrument) have t be used fr any PCR applicatin f ur kits. PIKA Weihenstephan GmbH des assume n respnsibility fr the lawfully use f this kit; this respnsibility lies expressly and slely at the user. The prcess f plymerase chain reactin is cvered by several patents. Fr cmmercial use, licensing by either f the cmpanies Rche and/r Applied Bisystems is needed. "N license under these patents t use the PCR prcess is cnveyed expressively r by implicatin t the purchaser by the purchase f this prduct. Further infrmatin n purchasing licenses t practice the PCR prcess may be btained by cntacting the Directr f Licensing at Rche Mlecular Systems, Inc., 1145 Atlantic Avenue, Alameda, Califrnia " # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 11 f 12

12 Develped and Manufactured by PIKA Weihenstephan GmbH Raiffeisenstrasse 31A Pfaffenhfen GERMANY +49 (0) (0) # e Fr everyne Detectin Kit Y Dekkera (Brettanmyces) sp. Screening User Guide page 12 f 12