foodproof StarPrep Two Kit Order No. S Quick Reference Procedure for Legionella

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1 foodproof StarPrep Two Kit Order No. S Quick Reference Procedure for Legionella Version 1, November 2017 A. Kit Contents / Storage and Stability Product Content Storage S container with 42 ml Lysis Buffer and a magnetic stir bar 15 to 25 C Kit for 192 isolations. The components of the foodproof StarPrep Two Kit are guaranteed to be stable through the expiration date printed on the label when stored at 15 to 25 C. B. Additional Equipment Required Standard tabletop microcentrifuge suitable for 1.5 ml tubes capable of a g centrifugal force Centrifuge suitable for 5 15 ml tubes and capable of a g centrifugal force Vortex Genie 2 from Scientific Industries Inc. (Order No. D ) with Large Ampule/Tube Attachment adapter (Order No. Z ) or Horizontal 15 ml Tube Holder (Order No. Z ) Unit for mechanical cell disruption suitable for working with 1.5 ml reaction tubes (e.g., Disruptor Genie from Scientific Industries Inc.) Filtration unit suitable for 47 mm filter membranes Heating Unit Magnetic Stirrer Tubes 5 ml, 58 x 15mm, Order No. Z PES membrane filters (e.g. Pall, 0.45 µm pore size, 47 mm, plain #60173; Order No. Z ) foodproof D-Light, Order No. D Reagent D, Order No. A Rinse Buffer, Order No. A All instruments and equipment can be purchased from BIOTECON Diagnostics. C. Before you Begin / Isolation Procedure Prepare filtration equipment and samples according to general laboratory procedures. The Lysis Buffer is optimized for the preparation of various types of sample material. The quality of the DNA obtained with the Lysis Buffer is suitable for any PCR application. In order to avoid cross-contamination use filter tips. Follow all universal safety precautions governing work with biohazardous materials (e.g., wear lab coats and gloves at all times). Properly dispose of all contaminated materials, decontaminate work surfaces, and use a biosafety cabinet whenever aerosols might be generated. To avoid foam formation of the Lysis Buffer, do not shake the container up and down. Mix thoroughly while pipetting the buffer for sample preparation. For mixing, use a magnetic stirrer at 350 rpm to move the stir bar in the container. Warm the heating unit to C. Thaw the Reagent D. 1

2 D. Procedure A This protocol is intended for quantification of Legionella in water samples using 47 mm filter disks. Note: For valid results, strictly adhere to the protocol below. Only use the filter material, tubes and Rinse Buffer specified by BIOTECON Diagnostics. Omit steps [7] [9] if removal of dead bacteria or residual DNA is not desired. Step Action Volume Time/g Time/Temp. 1 Add 1000 µl Rinse Buffer to a 5 ml tube µl 2 Filter sample. Write down the filtration volume. Note: Conduct filtration according to your laboratory s specifications. Acid or heat treatment of the filter is not necessary for real-time PCR analysis. 3 Transfer the filter paper to a 5 ml tube with the top side (filter cake) facing inwards. Note: Pick up the filter disk at opposing sides using two sterile forceps. Gently roll the membrane into the shape of a cylinder until it fits into the 5 ml tube. Be careful not to crumple the filter disk or touch its top side! 4 Insert tubes into Large Ampule/Tube Attachment or Horizontal 15ml Tube Holder on a Vortex Genie 2. Vortex at full speed, then vertically turn the tubes by 180 and repeat. Note: The tubes should be balanced. In case of processing an odd number of samples, add a 5 ml tube filled with 1ml distilled water as counterweight. 5 Centrifuge. Note: Use a centrifuge suitable for 5 15 ml tubes. If necessary, centrifugation forces should be calculated according to the manual of the centrifuge used. 6 Transfer 700 µl sample to a 1.5 ml transparent reaction tube. Note: In case recovery of 700 µl cannot be achieved, write down the approx. recovered volume and take into account when calculating the bacterial load of the sample. 7 Add 300 µl Reagent D. Mix by pipetting up and down for 3-5 times or brief vortexing. Note: Proceed immediately with the following steps of the protocol. Avoid extended exposure to light. For optimal efficiency, the mixing has to be complete. 8 Incubate in the dark at room temperature. Note: Incubate in the foodproof D-Light unit. 9 Exposure to light in the foodproof D-Light unit. Note: If you use a high-power halogen light bulb, place the reaction tubes approx. 20 cm from the light bulb on wet ice or in a cooling block. Samples must not freeze. 10 Centrifuge. Note: Use a centrifuge suitable for 1.5 ml tubes. 11 Remove the supernatant with a pipette immediately after centrifugation, discard, and inactivate appropriately. Note: Take care that the tip of the pipette in the reaction tube is on the opposite side of the pellet. 12 Transfer 150 µl Lysis Buffer to the reaction tube. Place the container of Lysis Buffer on the magnetic stirrer. Note: It is not recommended to use more than 192 reactions. There must retain some of the reagent in the container. Do not use anymore reagent after it has reached the minimal level mark on the container (the mark indicates the minimal allowed pipetting level while the stirrer is not in use). Hold the container while switching on the magnetic stirrer and during pipetting. Stir at 350 rpm to mix the Lysis Buffer gently and yield a homogeneous solution. This constant stirring avoids sedimentation of ingredients during pipetting. Open the Lysis Buffer container. Use a 1,000 µl filter tip to transfer 150 µl Lysis Buffer to the sample. Note: Pipet carefully and vertically along the container wall, approximately 0.5 cm above the bottom. suitable volume 2 x 15 sec 1,000 g 700 µl 300 µl 10 min 5 min 5 min at 8,000 g 150 µl 2

3 13 Place the tube in the Disruptor Genie for mechanical disruption and turn it on at maximum speed. 14 Incubate the suspension in a heating unit. 5 min at C 15 Carefully remove the reaction tube from the heating unit and allow the tube to sit. As the tube will be hot, use forceps for removal. 16 Mix by vortexing. 2 s 17 Centrifuge. Result: The supernatant now contains the extracted DNA and can be used directly for PCR. Note: Parts of the sediment may inhibit PCR and must not be used. 8 min C 13,000 g 3

4 E. Procedure B This protocol is intended for direct quantification of Legionella in water samples. Note: For valid results, strictly adhere to the protocol below. Note: Omit steps [2] [4] if removal of dead bacteria or residual DNA is not desired. Step Action Volume Time/g Time/Temp. 1 Transfer 700 µl sample to a 1.5 ml transparent reaction tube. Note: In case of very cloudy samples, only transfer 300 µl or less. Acid or heat treatment of the sample is not necessary for real-time PCR analysis. 2 Add 300 µl Reagent D. Mix by pipetting up and down for 5-10 times or brief vortexing. Note: Proceed immediately with the following steps of the protocol. Avoid extended exposure to light. For optimal efficiency, the mixing has to be complete. If sample volume had been reduced, add Reagent D at half the volume of the sample. E.g. 300 µl sample µl Reagent D. 3 Incubate in the dark at room temperature. Note: Incubate in the foodproof D-Light unit. 4 Exposure to light in the foodproof D-Light unit. Note: If you use a high-power halogen light bulb, place the reaction tubes approx. 20 cm from the light bulb on wet ice or in a cooling block. Samples must not freeze. 5 Centrifuge. Note: Use a centrifuge suitable for 1.5 ml tubes. 6 Remove the supernatant with a pipette immediately after centrifugation, discard, and inactivate appropriately. Note: Take care that the tip of the pipette in the reaction tube is on the opposite side of the pellet. 7 Transfer 150 µl Lysis Buffer to the reaction tube. Place the container of Lysis Buffer on the magnetic stirrer. 700 µl (or appropriate volume) 300 µl (or appropriate volume) 10 min 5 min 5 min at 8,000 g 150 µl Note: It is not recommended to use more than 192 reactions. There must retain some of the reagent in the container. Do not use anymore reagent after it has reached the minimal level mark on the container (the mark indicates the minimal allowed pipetting level while the stirrer is not in use). Hold the container while switching on the magnetic stirrer and during pipetting. Stir at 350 rpm to mix the Lysis Buffer gently and yield a homogeneous solution. This constant stirring avoids sedimentation of ingredients during pipetting. Open the Lysis Buffer container. Use a 1,000 µl filter tip to transfer 150 µl Lysis Buffer to the sample. Note: Pipet carefully and vertically along the container wall, approximately 0.5 cm above the bottom. 8 Place the tube in the Disruptor Genie for mechanical disruption, and turn it on at 8 min maximum speed. 9 Incubate the suspension in a heating unit. 5 min at C 10 Carefully remove the reaction tube from the heating unit, and allow the tube to sit. As the tube will be hot, use forceps for removal. 11 Mix by vortexing. 2 s 12 Centrifuge. Result: The supernatant now contains the extracted DNA and can be used directly for PCR. Note: Parts of the sediment may inhibit PCR and must not be used C 13,000 g 4

5 F. Procedures for identification of pure cultures The following protocol describes the DNA isolation from samples with a high amount of viable target organism (e.g., liquid cultures or colonies from agar plates). This protocol is suitable for identification of L. pneumophila or serotyping of L. pneumophila serogroup 1 from single specimens. Step Action Volume Time/g Time/Temp. 1 Transfer 150 µl Lysis Buffer to the reaction tube. Place the container of Lysis Buffer on the magnetic stirrer. Note: It is not recommended to use more than 192 reactions. There must retain some of the reagent in the container. Do not use anymore reagent after it has reached the minimal level mark on the container (the mark indicates the minimal allowed pipetting level while the stirrer is not in use). Hold the container while switching on the magnetic stirrer and during pipetting. Stir at 350 rpm to mix the Lysis Buffer gently and yield a homogeneous solution. This constant stirring avoids sedimentation of ingredients during pipetting. Open the Lysis Buffer container. Use a 1,000 µl filter tip to transfer 150 µl Lysis Buffer to the sample. 150 µl Note: Pipet carefully and vertically along the container wall, approximately 0.5 cm above the bottom. 2 For liquid cultures Shake the enrichment culture gently and let settle for 5-10 min. Transfer 25 µl sample (supernatant) to the 1.5 ml reaction tube containing the Lysis Buffer min 25µl For colonies Transfer a small part of the colony with a suitable tool (e.g., inoculating needle) to the 1.5 ml reaction tube containing the Lysis Buffer. 3 Place the tube in the Disruptor Genie for mechanical disruption, and turn it on at 8 min maximum speed. 4 Incubate the suspension in a heating unit. 5 min at C 5 Carefully remove the reaction tube from the heating unit, and allow the tube to sit. As the tube will be hot, use forceps for removal. 6 Mix by vortexing. 2 s 7 Centrifuge. Result: The supernatant now contains the extracted DNA and can be used directly for PCR. Note: Parts of the sediment may inhibit PCR and must not be used C 13,000 g 5

6 G. Storage of samples If you want to Then Continue Stop Use the extracted DNA directly. Strictly avoid transferring fractions of the sediment to the PCR reaction, because this might cause PCR inhibition. Store the DNA at -15 to -25 C for later analysis. After thawing mix short by vortexing and centrifuge again at 13,000 g for 5 min. Note: The sample is not purified. Proteins, RNA, and other materials remain in the sample. Longterm storage or archival of prepared DNA samples is not recommended. H. Troubleshooting Problem Possible cause Recommendation DNA sample inhibits PCR Sample contains too many PCR inhibitors. Some of the centrifugation pellet transferred to the PCR. Reagent D carry-over. Dilute the DNA extract (e.g. 1:5). Repeat with reduced sample volume (e.g. 300µl). Use the top of the supernatant as PCR template. Always centrifuge the DNA sample before performing PCR. Completely remove the supernatant after Reagent D treatment steps without touching the pellet. Low DNA yield Inappropriate storage. Store at C. Lid of the reaction tube opens during or after heating No or insufficient beads in the reaction. Water sample contains substances that reduce the DNA extraction efficiency. Pellet resuspension incomplete. Reaction tube not firmly closed. Use correct stirring settings. Do not pipette more than 192 reactions. Reduce the water sample volume where applicable. Prolong the resuspension time. Ensure that all reaction tubes are firmly closed before heating. Use lid clips for closing the tubes. Use a heating unit that enables removal of the tube without contact with the tube. For further information please refer to: BIOTECON Diagnostics GmbH Hermannswerder Potsdam Germany Phone +49 (0) Fax +49 (0) bcd@bc-diagnostics.com S (1) 6