Instruction Protocol M4BD ()
Contents
1. Spin a 96 Deep Well Plate 1 min at 1000 g and remove seal Add 0 5 ml milk sample to each of the wells Cover wells with sealing tape Incubate the Deep Well Plate at 37 C for 10 min Spin Deep Well Plate with milk at 37 C and 5000xg for 5 min and discard sealing tape Remove supernatants from the top with tips connected to a vacuum system Be careful not to touch or remove the bacterial pellets Add 1 mL Wash Buffer to each tube Cover with sealing tape Spin at 5000xg and at 37 C for 5 min and discard sealing tape Remove supernatants from the top with tips connected to a vacuum system Be careful not to touch or remove the bacterial pellets It is important to remove the supernatant completely Prepare fresh Lysis l Mix by adding 3 uL Mix I additive to 27 uL Lysis buffer l and mix Lysis l Mix 1 reaction 100 reactions Lysis Buffer l 27 uL 2700 uL Mix additive 3 uL 300 uL TOTAL 30 uL 3000 uL 7 Add 30 uL Lysis l Mix to each pellet and cover with sealing tape Vortex the Deep Well plate 10 seconds and spin 20 seconds at 1000xg to bring Lysis mix to the tube bottoms 8 Remove caps from 96 well plate with clear 0 2 mL tubes 9 Use an 8 channel pipette with filter tips for transfer of 30 uL from each well of the Deep Well Plate into the corresponding tubes in the 96 well plate clear 0 2 mL Close tubes with the caps Avoid cross contamination Use one new tip per tube 10 Incubate the 96 well pl2. DNA DIAGNOSTIC e Staphylococcus aureus e Streptococcus agalactiae Mastit 4BD e Streptococcus uberis e Mycoplasma bovis e Mycoplasma species e Streptococcus dysgalactiae e Penicillin resistance gene p lactamase from Staphylococci e Coagulase Negative Staphylococci Cat No M4BD USER MANUAL Revision 2015 08 15 TABLE OF CONTENTS 1 PRINCIPEE OF THE TEST ob o Oo iE onm E nn teinte bait nemen 2 KIT COMPONENTS AND STORAGE 25 iecizesessu tccocka acce ue stud qux eme en zo ve ek tae cute era qeu Evae RUN ue EON ERE Du ea 3 REQUIRED EQUIPMENT sinistres dnengertee eoe eer ete er bao de eoe Eee redo ro bad ie anale ues iii neta Pub ee Ai PROTOCOL ceia otii remeses ates cec d ca LUE 5 INTERPRETATION OF TEST RESULTS eene eene enne nennen nenne renes ener nenne nene nenne nennen nennen Trademarks Cy5 is a registered trade mark of GE Healthcare Mx3005P Stratagene and Agilent are registered trademarks of Agilent Technologies Inc Black Hole Quencher BHQG CAL Fluoor and Quasar dye technology incorporated in this product are used under licensing agreement with Biosearch Technologies Inc and protected by U S and world wide patents issued or in application 1 PRINCIPLE OF THE TEST The Mastit 4 test with Cat No M4BD can be used for screening of 384 milk samples for eight targets Staphylococcus aureus Mycoplasma species Streptococcus agalactiae Streptococcus dysgalactiae Streptococcus uberis Penicillin resi
3. ains three boxes one for storage at room temperature and two for storage at 20 C Box for storage at room temperature contains e Four sealed Deep Well Plates each with 96 wells containing a solution e Four bottles with Wash Buffer 100 mL each e Four 96 Well Plates 0 2 mL clear tubes with caps e 12 pieces of sealing tapes for Deep Well plates e Eight 96 cap mats for the qPCR 96 Well Plates Box for storage at 20 C contains e Two bottles with Lysis Buffer l 5400 uL each e Two yellow capped tubes with Mix Additive for Lysis Buffer l 600 uL each e Four sealed 96 Well Plates 0 2 mL white tubes with qPCR Master Mix M4B Box Il for storage at 20 C contains e Four sealed 96 Well Plates 0 2 mL white tubes with qPCR Master Mix M4D REQUIRED EQUIPMENT Centrifuge for 96 well plates centrifuge must be capable of running at 5000xg at 37 C Pipettes and sterile filter tips Incubation oven at 37 C Plate washer vacuum system to aspirate supernatant from Deep Well Plates Biorad DW40 can be used PCR instrument for 37 C and 95 C incubations during lysis Alternatively the qPCR machine can be used qPCR instrument Stratagene Mx3005 with filters for ATTO 440nm 492nm FAM 492nm 516nm HEX 535nm 555nm ROX 585nm 610nm CY5 635nm 665nm 0 5 mL milk Lysis 37 C 20 min Add template qPCR Interpretation Spin Wash Lysis 95 C 15 min Spin 4 PROTOCOL DNA purification 1
4. ate at 37 C for 20 min 11 Incubate the 96 well plate at 95 C for 15 min 12 Cool the 96 well plate on ice for 5 min Note The incubation at 37 C and 95 C can be done using a PCR instrument programmed 37 C for 20 min 95 C for 15 min 4 C for 5 min 13 Centrifuge the 96 well plate at 5000xg for 5 min at room temperature Note it is important to use a 96 tube support for the 96 well plate during centrifugation 14 Carefully remove the caps from the 96 well plate Use an 8 channel pipette with filter tips to transfer 5 uL of each aqueous phase directly to the corresponding tubes of the 96 well plate with qPCR Master Mix in step 18 15 The remaining purified DNA can be stored at 20 C for long time storage qPCR analysis 16 Take a 96 Well Plate with qPCR Master Mix white 0 2 mL tubes from 20 C place on ice for five minutes and spin 20 seconds at 1000xg to bring qPCR Master Mix to the tube bottoms 17 Discard the seal from the 96 Well Plate with qPCR Master Mix and place the 96 well plate on ice 18 Transfer 5 uL purified DNA from step 14 to each of the corresponding tubes in the 96 well plate containing 15 uL qPCR Master Mix Note It is important to keep Master Mix on ice while loading template and to run the qPCR within 15 min 19 Carefully close the qPCR tubes with a new optical lid Spin the tubes briefly before transfer of the 96 Well Plate qPCR reactions to the qPCR instrument Note it is important to k
5. eep the optical lids clean 20 Repeat step 16 19 with M4L qPCR Master Mix Instrument settings for the MX3005P 21 Filter Gain Settings ATTO FAM HEX ROX CY5 1x 2x 1x 1x 1x Note If the raw data signal R is lower than 5000 for a color at the cycles 7 11 then increase the filter gain setting during the next runs resulting in a raw data signal R of 5000 35000 22 Threshold Fluorescence Select Background Based Threshold to cycles 7 11 and set the Sigma multiplier to 10 23 Baseline Correction Select Adaptive baseline 24 Activate the following filters ATTO FAM HEX ROX CY5 25 Run qPCR with the following cycling parameters Temperature Time Cycles Comment 95 C 1 min 1 Heat activation 95 C 5 sec Amplification 40 Read fluorescence for for ATTO FAM HEX 60 C 25 sec ROX CY5 at the end of each extension step 5 INTERPRETATION OF TEST RESULTS Generate amplification plots showing e Thermal cycles on the X axis e Background subtracted fluorescence dR on the Y axis e Ct values are calculated automatically by the instrument The Stratagene MxPro software should be set to Adaptive Baseline Correction and Background based Threshold using cycle 7 11 and a sigma multiplier of 10 e Samples with Ct values below 37 and S shaped amplification curves are considered as true positive e Samples with Ct values above 37 may be a result of unspecific amplification fal
6. es and the IAC Streptococcus uberis CY5 IAC ATTO Streptococcus agalactiae ROX IAC ATTO Staphylococcus aureus HEX IAC AT TO Mycoplasma bovis FAM IAC ATTO 10 Mycoplasma species CY5 IAC ATTO Staphylococci B lactamase ROX IAC ATTO 11 Coagulase Negative Staphylococci HEX IAC ATTO Streptococcus dysgalactiae FAM IAC ATTO 12 DNA DIAGNOSTIC e Staphylococcus aureus e Streptococcus agalactiae e Streptococcus uberis Mastit 4BD ME Mycoplasma bovis e Mycoplasma species e Streptococcus dysgalactiae e Penicillin resistance gene p lactamase from Staphylococci e Coagulase Negative Staphylococci For more information contact DNA Diagnostic A S Voldbjergvej 16 8240 Risskov Denmark Tel 45 8732 3050 E mail info dna diagnostic com www dna diagnostic com DNA Diagnostic A S was established in 1992 DNA Diagnostic A S is an ISO 9001 and ISO 13485 certified developer manufacturer and worldwide supplier of PCR based in vitro diagnostic kits Cat No M4BD USER MANUAL
7. se positive The test should be consider negative An IAC amplification signal in the ATTO filter should be present in all reactions The IAC Ct value must be between 27 and 32 for the test to be valid Ct above 32 is only accepted when the reaction is highly positive low Ct value for one of the bacteria in the test INTERPRETATION TABLE Master qPCR target mix Streptococcus uberis Filter Positive Negative Ct Retest Ct Streptococcus agalactiae Staphylococcus aureus Mycoplasma bovis IAC Mycoplasma species 232 or absent Penicillin resistance gene B lactamase from Staphylococci Coagulase Negative Staphylococci Streptococcus dysgalactiae IAC gt 32 or absent If the IAC Ct is 532 or absent the purified DNA should be diluted 10 times and a new qPCR reaction should be analyzed using this diluted DNA A dilution of 10 times will theoretical move Ct values 3 3 higher than undiluted dR 4 Threshold line 0 10 20 30 40 Cycles Amplification plot with X axis showing cycle number and Y axis showing background subtracted fluorescence The threshold line is used to find the Ct value Ct is the intersection between the amplification curve and the threshold line The threshold line is calculated automatically by the instrument In this example Ct 24 4 Eight examples of amplification plots each positive for one bacteria speci
8. stance gene B lactamase from Staphylococci Mycoplasma bovis Coagulase Negative Staphylococci CNS Mastit 4 is the name of a family of fast sensitive and easy to use tests for detection of four mastitis causing bacteria species in each qPCR reaction Mastit 4 testing involves an easy to do DNA extraction from 0 5 mL milk samples followed by a fast qPCR reaction Test results can be acquired in 3 hours with very little hands on time The Mastit 4 tests can be used on bulk tank milk or individual milk samples The samples can be preserved or non preserved Each of the two qPCR tests in M4BD use approximately 1 6 of the purified DNA as template The remaining DNA can be used for testing with other Mastit 4 variants or stored at 20 C for later use Each qPCR reaction contains four sets of primers and fluorescence probes for specific detection of four bacteria A fifth set of primers and probe detects an internal amplification control IAC The qPCR instrument generates an amplification plot and Ct values Cycle at threshold Each of the four bacteria probes emit a specific light color enabling the identification of bacteria present in the sample The tests use the standard 96 well plate format and ready to use reaction mixes The test requires only pipettes a vacuum aspirator a centrifuge a thermal heating block and a qPCR instrument 2 KIT COMPONENTS AND STORAGE The Mastit 4BD kit contains material for testing 4x96 samples One kit cont
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