First-Alicyclobacillus multiplex PCR kit

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1 First-Alicyclobacillus multiplex PCR kit Art..: TPABM 0050 R (Biorad, Agilent Mx3500p, Rotorgene ) (Version 09/13) 1. Intended use Qualitative detection and identification of Alicyclobacillus spp., Alicyclobacillus acidocaldarius and Alicyclobacillus acidoterrestris 2. Test principle The TaqMan realtime PCR is based on Hotstart-PCR and sequence-specific dual labelled probes (FAM/DQ; CY5/DQ; HEX/DQ) which, when accurately hybridised, emit a measurable fluorescent signal of a defined wavelength in the extension phase. The increase of signal is continuously measured in a realtime PCR detection instrument. To avoid false negative PCR-results an Inhibition control (ROX/DQ) is amplified together in one reaction vessel with the specific sequence. The system contains dutp. Optional: the use of Uracil-N-Glycosylase will eliminate any contamination with Uracil containing amplicons from former PCRs (the enzyme is not part of this kit). 3. Kit contents The TPABM 0050 kit contains sufficient reagents for 50 PCR reactions: 1 x Premix TPABM white cap 1 x TPABM mix (freeze-dried) dark vial, red cap 1 X Inhibition Control-DNA (freeze-dried ) black cap 1 x dd H 2 0 (for solving dried reagents) colourless cap 1 x Control-DNA (freeze-dried) yellow cap (contains A. acidocaldarius and A. acidoterrestris DNA) - 8 -

2 4. Storage conditions The TPABM mix, the Inhibition Control-DNA and the Control-DNA are freezedried, they have to be solved in ddh 2 O prior to use (see 6.1). The freeze-dried and solved PCR reagents should be stored at 2 8 C (35 46 F). All reagents are stable for 6 months at 2 C 8 C (35 46 F). The Premix should be stored at -18 C (-0, 4 C). Stored at 2 C 8 C (35 46 F) the Premix is stable for two weeks. The TPABM mix contains the light sensitive hybridisation probes and should be handled light protected. 5. Materials required but not provided 5.1. Instruments: Realtime machine with at least 4 channels Centrifuge for 1,5 2,0 ml tubes Swing-bucket centrifuge for multiwell plates / stripes Suitable pipettes for PCR: 0, µl Vortex 5.2. Reagents and plastic ware: sterile ddh 2 0 sterile reaction vessels 1,5 2,0 ml PCR plates / stripes sterile filter tips optional: Uracil N-Glycosylase (0,01 U/µL added to the PCR reaction mix) 6. Test procedure 6.1. PCR-Setup Preparations before first use: When using the kit for the first time, the freeze-dried kit components have to be shortly centrifuged and carefully resolved: add 80 µl sterile ddh 2 0 to the freeze-dried TPABM mix add 27 µl sterile ddh 2 0 to the freeze-dried Inhibition Control-DNA add 55 µl sterile ddh 2 0 to the freeze-dried Control-DNA after 30 minutes mix well Before every use thoroughly mix all PCR components and centrifuge briefly

3 PCR-reaction Setup: PCR-Components volume (µl) Premix TPABM 13,0 TPABM mix 1,5 Inhibition Control-DNA 0,5 Sample-DNA 5,0 Total volume 20,0 Prepare a master mix by mixing Premix, TPABM mix and Inhibition Control-DNA. 1. Multiply said volumes with the number of PCR preparations including controls (positive control, negative control, extraction control), taking into account pipette reserves of approximately 5-10 %. 2. Pipette 15 µl of the PCR-master mix into the PCR-tubes or multiwell plate, making sure that, prior to the first filling, the tip of the pipette has been moistened. 3. Add the using a fresh tip with each DNA filling and mix it with the master mix at the same time, (with DNA volume less than 5 µl add ddh 2 O to the end volume of 20 µl per reaction). Add 5 µl of the Control-DNA, 5 µl of the extraction control and 5 µl of ddh 2 O for the negative control reaction. 4. Seal the plate properly with a Sealing Foil by pressing it firmly to the plate surface using the scraper. Centrifuge the plate. 5. Place the multiwell plate / PCR-tubes into the loading frame of the loader. Work swiftly to avoid warming up and keep away from light. 6.2 PCR-Program Realtime machine with PCR-tubes / multiwell plates Optional: UNG-Incubation Step Time Temp. Acquisition Status Mode Initial denaturation of DNA 10 min 95 C none Hold Denaturation 15 sec 95 C none Cycle 40-45x Annealing/ Elongation 25 sec 60 C single Cooling 15 sec 40 C none Hold Ramp Rate Maximal

4 7. Interpretation of results The evaluation has to be made according to the data analysis program recommended by the realtime instrument manufacturer. Detection of Alicyclobacillus spp: HEX-channel Detection of Alicyclobacillus acidoterrestris: FAM-channel Detection of Alicyclobacillus acidocaldarius: CY5-channel Detection of Inhibition control: ROX-channel A sample is Alicyclobacillus spp positive, if there is a detectable fluorescence increase in the HEX-channel and the negative controls show no amplification. The positive controls should have a positive fluorescence signal. The inhibition control in the ROX-channel may be positive or negative (depending on the amount of DNA or inhibitors in the sample reaction), in the negative controls it must be positive. A sample is Alicyclobacillus acidoterrestris positive, if there is a detectable fluorescence increase in the FAM-channel and the negative controls show no amplification. The positive controls should have a positive fluorescence signal. The inhibition control in the ROX-channel may be positive or negative (depending on the amount of DNA or inhibitors in the sample reaction), in the negative controls it must be positive. A sample is Alicyclobacillus acidocaldarius positive, if there is a detectable fluorescence increase in the CY5-channel and the negative controls show no amplification. The positive controls should have a positive fluorescence signal. The inhibition control in the ROX-channel may be positive or negative (depending on the amount of DNA or inhibitors in the sample reaction), in the negative controls it must be positive. A sample is negative, if there is no detectable fluorescence increase in the different channels and the positive controls have a positive fluorescence signal. The negative controls show no amplification. The Inhibition-control in the ROX-channel must be positive in the sample and in the negative controls, a false negative result due to inhibitory effects is then excluded

5 analysis flowchart Channels: HEX, FAM, CY5, ROX Fluorescence increase with control-dna and no fluorescence increase with negative control?. PCR not successful! HEX-channel FAM-channel CY5-channel ROX-channel (Inhibition control) sample PCR inhibited! Alicyclobacillus spp. DNA! Alicyclobacillus acidoterrestris DNA! Alicyclobacillus acidocaldarius DNA! no Alicyclobacillus spp. DNA Alicyclobacillus acidoterrestris DNA Alicyclobacillus acidocaldarius DNA te: The polymerase-chain reaction (PCR) is protected by patents and requires a licence from Hoffmann-LaRoche Inc.. The provided product does not authorise the purchaser for the commercial use of this method. GEN-IAL makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. If any materials are defective, GEN-IAL will provide a replacement product. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. GEN-IAL shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product