Hygiena KIT230187 Vibrio Detection LyoKit Instruction Manual

Hygiena KIT230187 Vibrio Detection LyoKit

Specifications

• Product No.: KIT230187
• Approval: LICENSE NUMBER 102004
• Revision: A, September 2023

Product Information

Overview

• The kit is designed for 96 reactions.

Applicability

• The lysis buffer is optimized for the preparation of enrichment cultures of various types of sample material, including meat and sprouts. The quality of the DNA obtained with the lysis buffer is suitable for any PCR application.

Kit Contents

• The kit includes a container with 21 mL lysis buffer 1 and a magnetic stir bar.

Instructions

• Equipment needed includes a centrifugal force device (e.g., Micro-Star 21), a heating unit for 1.5 mL tubes, a vortex mixer, and a magnetic stirrer. Reagents required include sterile 0.85% NaCl solution.

Safety and Preparations

• Ensure to use only recommended materials to guarantee the method’s robustness.

Workflows

• Extraction Procedure A
• Detailed instructions for Procedure A.

Extraction Procedure B

• Detailed instructions for Procedure B.

Extraction Procedure High Target Amount

• Detailed instructions for extracting high target amounts.

Extraction Procedure: Colonies

• Instructions specific to extracting from colonies.

Troubleshooting

• Guidelines for common issues during extraction.

Support

• Contact information for customer support.

Additional Information

• Additional general information about product usage.

Reference Number

• Reference number for further inquiries.

Change Index

• Index for tracking revisions and changes in instructions.

FAQ

Q: What is the storage condition for the kit?

• A: The storage conditions are…

Q: How many reactions can be performed with the kit?

• A: The kit is designed for 96 reactions.

OVERVIEW

• The foodproof® StarPrep® Three Kit is designed for the rapid preparation of DNA from Gram-negative bacteria like Shiga toxin-producing E. coli (STEC) for direct use in PCR.
• In less than 30 minutes, preparation with this lysis buffer yields PCR template DNA from enrichment cultures. The extracted DNA can be used directly in any PCR application.
• The StarPrep Three Lysis Buffer eliminates the need for hazardous organic extractions or chaotropic agents.
• The entire DNA preparation can be performed in a single tube, minimizing handling steps and exposure to hazardous material.
• The reduced number of handling steps saves time and eliminating DNA-containing extract transfer steps minimizes the risk of cross-contamination.

General Information

Number of Reactions

• The kit is designed for 96 reactions.

Storage Conditions

• Store at 15 to 25 °C.
• The components of the food-proof StarPrep Three Kit are guaranteed to be stable through the expiration date printed on the label.

Applicability

• The lysis buffer is optimized for the preparation of enrichment cultures of various types of sample material, including meat and sprouts.
• The sample volume varies depending on which matrix is being tested. The quality of the DNA obtained with the lysis buffer is suitable for any PCR application.

Kit Contents

• A schematic representation of the foolproof StarPrep Three Kit with all its components.

KIT230187

INSTRUCTIONS

• This section provides all information for a seamless DNA extraction from a variety of matrices.

Required Material

• Most of the required equipment and reagents are available through Hygiena®.
• Please contact us for further information.
• It is highly recommended to only use the materials described below to guarantee the robustness of the method.

Equipment

• Standard tabletop microcentrifuge capable of a 13,000 × g centrifugal force
  • e. g., Micro-Star 21
• Heating unit suitable for 1.5 mL tubes capable of a temperature range of 95 to 100 °C
  • e.g., AccuBlock™ – Labnet with heating block
• Vortex mixer
  • e. g., Vortex-Genie® – Scientific Industries
• Magnetic stirrer
  • e. g., Color squid IKAMAG® – IKA®-Werke

Reagents

• Sterile 0.85 % NaCl solution Not provided
  • Only for procedure B (2.3.2)

Safety and Preparations

• Follow all universal safety precautions governing work with biohazardous materials, e.g., wear lab coats, gloves, and other personal protective equipment at all times. The assay should only be used by adequately trained personnel.
• Properly dispose of all contaminated materials, and clean and decontaminate work surfaces with an appropriate disinfectant of choice (e.g., sodium hypochlorite solution) before and after use as part of aseptic techniques.
• Use a biosafety cabinet whenever aerosols are generated.
• In addition to cleaning workstations, work areas should be separated for the following: media preparation, sample preparation, and pathogen detection. Laboratory equipment like pipettes or tubes must not circulate between workstations.
• When working with enrichment cultures, filter laboratory bags should be used to minimize particulates, and shaking the enrichment bag or collecting large food fragments should be avoided.
• For fatty foods, collect the sample just below the fat layer. Never reuse kit disposables and always change serological pipettes and pipette tips between samples.
• All contaminated and potentially infectious material, like enrichment cultures or food samples, should be autoclaved before disposal and eliminated according to local rules and regulations.
• For more information, e.g., proper disposal of unused chemicals, please refer to the appropriate safety data sheet (SDS).
• The SDS is available online at www.hygiena.com/sds.
• Always use filter tips to avoid cross-contamination.
• Mix thoroughly while pipetting the buffer for sample preparation.
• Set the heating unit to 95 to 100 °C.

Workflows

• The following procedures describe the DNA isolation from enrichment cultures. Compared to Extraction Procedure A, Extraction Procedure B includes an additional washing step, if the enrichment medium contains acriflavine or other fluorescent substances.
• The “High Target Amount” protocol describes the DNA isolation from enrichment cultures with a high amount of target organisms, and the “Colonies” protocol includes the DNA isolation from bacterial colonies.
• We offer an additional rapid protocol for colony confirmation in combination with the microproof® Suspension Buffer (Product No. KIT 2301 78. Please refer to the product instructions).

EXTRACTION PROCEDURE A

This protocol describes the DNA isolation from enrichment cultures.

1. SHAKE SAMPLE
   • Shake the enrichment culture gently and let the suspension settle for 5 to 10 min.
2. ADD SAMPLE
   • Transfer 100 µL sample (supernatant enrichment culture, 20 to 24 h enrichment time point) to a 1.5 mL reaction tube.
   • Note: For 25 g meat sample enrichment cultures with 8 to 20 h enrichment time, please use 500 µL For 375 g meat sample enrichment cultures with 12 to 20 h, please use 500 µL.
3. CENTRIFUGE
   • 5 min at 8,000 x g.
   • Note: If enrichment cultures are totally clear, centrifugation at ≥ 13,000 × g is recommended.
4. REMOVE SUPERNATANT
   • Discard liquid with a pipette immediately after centrifugation and inactivate appropriately.
   • Note: Take care that the tip of the pipette is on the opposite side of the pellet during pipetting.
5. ADD LYSIS BUFFER
   • Transfer 200 µL StarPrep Three Lysis Buffer to the sample tube.
   • Note: Use a magnetic stirrer (low speed) or gently shake the bottle with the lysis buffer for a short while.
6. MIX
   • Vortex or mix by pipetting up and down until pellet has completely resuspended.
7. INCUBATE
   • 10 min at 95 to 100 °C in a heating unit.
   • Carefully remove the reaction tube from the heating unit and allow the tube to sit for 1 min at 15 to 25 °C.
8. MIX
   • Vortex for 2 sec.
9. CENTRIFUGE
   • 2 min at 13,000 x g.

SUPERNATANT FOR DETECTION

• Use 25 µL of extract for the foolproof STEC LyoKits.
• Strictly avoid transferring fractions of the sediment to the PCR reaction, because this might cause PCR inhibition.
• For later analysis, store DNA at -15 to -25 °C.
• After thawing, mix briefly by vortexing and centrifuge at 13,000 × g for 2 min.

EXTRACTION PROCEDURE B

The following protocol describes the DNA isolation from 100 µL of enrichment culture containing acriflavine, e.g., mTSB + A (ISO/TS 13136) and mBPWp plus Acriflavin-Cefsulodin-Vancomycin (ACV) Supplement (BAM Chapter 4A).

1. SHAKE SAMPLE
   • Shake the enrichment culture gently and let the suspension settle for 5 to 10 min.
2. ADD SAMPLE
   • Transfer 100 µL sample (supernatant enrichment culture) to a 1.5 mL reaction tube.
3. CENTRIFUGE
   • 5 min at 8,000 x g.
   • Note: If enrichment cultures are totally clear, centrifugation at ≥ 13,000 × g is recommended.
4. REMOVE SUPERNATANT
   • Discard liquid with a pipette immediately after centrifugation and inactivate appropriately.
   • Note: Take care that the tip of the pipette is on the opposite side of the pellet during pipetting.
5. ADD BUFFER
   • Resuspend pellet in 1 mL 0.85 % NaCl.
6. CENTRIFUGE
   • 5 min at 8,000 x g.
   • Note: If enrichment cultures are totally clear, centrifugation at ≥ 13,000 × g is recommended. Use e.g., latex beads (Sigma, Catalog-No. LB11; add 10 µL of the suspension, 1:10 in distilled water) to increase efficiency and yield a visible pellet.
7. REMOVE SUPERNATANT
   • Discard liquid with a pipette immediately after centrifugation and inactivate appropriately.
   • Note: Take care that the tip of the pipette is on the opposite side of the pellet during pipetting.
8. ADD LYSIS BUFFER
   • Transfer 200 µL StarPrep Three Lysis Buffer to the sample tube.
   • Note: Use a magnetic stirrer (low speed) or gently shake the bottle with the lysis buffer for a short while.
9. MIX
   • Vortex or mix by pipetting up and down until the pellet has completely resuspended.
10. INCUBATE
    • 10 min at 95 to 100 °C in a heating unit.
    • Carefully remove the reaction tube from the heating unit and allow the tube to sit for 1 min at 15 to 25 °C.
11. MIX
    • Vortex for 2 sec.
12. CENTRIFUGE
    • 2 min at 13,000 x g.

SUPERNATANT FOR DETECTION

• Use 25 µL of extract for the foolproof STEC LyoKits.
• Strictly avoid transferring fractions of the sediment to the PCR reaction, because this might cause PCR inhibition.
• For later analysis, store DNA at -15 to -25 °C.
• After thawing, mix briefly by vortexing and centrifuge at 13,000 × g for 2 min.

EXTRACTION PROCEDURE “HIGH TARGET AMOUNT”

This protocol describes the DNA isolation from enrichment cultures with a high amount of the target organisms.

1. SHAKE SAMPLE
   • Shake the enrichment culture gently and let the suspension settle for 5 to 10 min.
2. ADD LYSIS BUFFER
   • Transfer 200 µL lysis buffer to a 1.5 mL reaction tube.
   • Note: Use a magnetic stirrer (low speed) or briefly shake the bottle gently before pipetting the lysis buffer to avoid sedimentation of ingredients.
3. ADD SAMPLE
   • Transfer 50 µL sample (enrichment culture supernatant) to the reaction tube containing the lysis buffer and mix briefly.
4. INCUBATE
   • 10 min at 95 to 100 °C in a heating unit.
   • Carefully remove the reaction tube from the heating unit and allow the tube to sit for 1 min at 15 to 25 °C.
5. MIX
   • Vortex for 2 sec.
6. CENTRIFUGE
   • 2 min at 13,000 x g.

SUPERNATANT FOR DETECTION

• Use 25 µL of extract for the foolproof STEC LyoKits.
• Strictly avoid transferring fractions of the sediment to the PCR reaction, because this might cause PCR inhibition.
• For later analysis, store DNA at -15 to -25 °C.
• After thawing, mix briefly by vortexing and centrifuge at 13,000 × g for 2 min.

EXTRACTION PROCEDURE: “COLONIES

For a rapid protocol for colony confirmation, please refer to our Suspension Buffer Product Instructions (Product No. KIT230178) or contact our support.

1. ADD LYSIS BUFFER
   • Transfer 200 µL lysis buffer to a 1.5 mL reaction tube.
   • Note: Use a magnetic stirrer (low speed) or briefly shake the bottle gently before pipetting the lysis buffer to avoid sedimentation of ingredients.
2. ADD PICKED COLONIES
   • Transfer a small part of the colony with a suitable tool.
   • (e.g., inoculating needle) to the reaction tube containing the lysis buffer and mix by gently swirling.
3. INCUBATE
   • 10 min at 95 to 100 °C in a heating unit.
   • Carefully remove the reaction tube from the heating unit and allow the tube to sit for 1 min at 15 to 25 °C.
4. MIX
   • Vortex for 2 sec.
5. CENTRIFUGE
   • 2 min at 13,000 x g.

SUPERNATANT FOR DETECTION

• Use 25 µL of extract for the foolproof STEC LyoKits.
• Strictly avoid transferring fractions of the sediment to the PCR reaction, because this might cause PCR inhibition.
• For later analysis, store DNA at -15 to -25 °C.
• After thawing, mix briefly by vortexing and centrifuge at 13,000 × g for 2 min.

Troubleshooting

broth.

Repeat DNA extraction with a reduced sample volume.

For very cloudy supernatants, a reduction of the sample volume might enhance DNA isolation efficiency.

DNA extract contains too many PCR inhibitors.| Dilute DNA extract, e.g., 1:10, or reduce the amount of extracted DNA, e.g., for LyoKits 5 µL + 20 µL PCR- grade H2O instead of 25 μl.
Some of the centrifugation pellets were transferred over to the PCR.| Always centrifuge the DNA sample before performing PCR.

Use the top of the supernatant as a PCR template.

Do not allow the filter tip to make contact with the pellet.

Supernatants are not completely removed.| Remove supernatants completely.
Low DNA yield.| Improper storage of kit components.| Store kit reagents at 15 to 25°C.
Enrichment culture contains substances that reduce the DNA extraction efficiency.| Perform a subcultivation or dilution, e.g., 1:10 in fresh enrichment broth.
The sample contains substances that reduce the DNA extraction efficiency.| Reduce the sample volume. Important note: this will also reduce sensitivity.
Not enough target organisms in the enrichment culture.| Prolong the incubation phase.
Enrichment culture is totally clear| Centrifuge at ≥ 13,000 × g.

Use latex beads (Sigma, Catalog-No. LB11; add 10 µL of the suspension, 1:10 in distilled water).

Pellet resuspension incomplete.| Improve resuspension by prolonged pipetting or vortexing.
Suboptimal reaction conditions.| Ensure proper heating conditions.

Verify the correct temperature of the heating block with a thermometer.

The lid of the reaction tube opens during or after heating.| The reaction tube is not firmly closed.| Ensure that all reaction tubes are firmly closed before heating.

Use lid clips to close the tubes properly.

Use a heating unit that enables the removal of the tubes without directly touching the tube lids.

Support

• If you have questions or experience any problems with our products, please contact us: at www.hygiena.com/support
• We aim to provide you with a solution as quickly and effectively as possible.
• We would also like you to contact us if you have any suggestions for improving the product or in case you would like to use our product for a different application. We highly value your feedback.

ADDITIONAL INFORMATION

Quality Control

• All products are regularly monitored by our quality control. You can find the certificate of analysis (COA) on our website.
• If you would like to carry out your quality control, you will find the analysis method described in the certificate.

Waste Disposal

• All contaminated and potentially infectious material, like enrichment cultures or food samples, should be autoclaved before disposal and eliminated according to local rules and regulations.
• For proper disposal of unused chemicals, please refer to the SDS.

Warranty and Disclaimer of Liability

Limited Warranty” and “Disclaimer of Liability”: Hygiena Diagnostics GmbH warrants that this product is free from defects in materials and workmanship through the expiration date printed on the label and only if the following are complied with:

1. The product is used according to the guidelines and instructions outlined in the product literature;
2. Hygiena Diagnostics GmbH does not warrant its product against any defects when: the defect is as a result of material or workmanship not provided by Hygiena Diagnostics GmbH; defects caused by misuse or use contrary to the instructions supplied, or improper storage or handling of the product;
3. All warranties of merchantability and fitness for a particular purpose, written, oral, expressed, or implied, shall extend only for one year from the date of manufacture. There are no other warranties that extend beyond those described on the face of this warranty;
4. Hygiena Diagnostics GmbH does not undertake the responsibility to any purchaser of its product for any undertaking, representation, or warranty made by any dealers or distributors selling its products beyond those herein expressly expressed unless expressed in writing by an officer of Hygiena Diagnostics GmbH;
5. Hygiena Diagnostics GmbH does not assume responsibility for incidental or consequential damages, including, but not limited to responsibility for loss of use of this product, removal or replacement labor, loss of time, inconvenience, expense for telephone calls, shipping expenses, loss or damage to property or loss of revenue, personal injuries or wrongful death;
6. Hygiena Diagnostics GmbH reserves the right to replace or allow credit for any modules returned under this warranty.

Trademarks

• foodproof®, microproof®, vetproof®, ShortPrep®, StarPrep®, RoboPrep® and LyoKit® are registered trademarks of Hygiena Diagnostics GmbH.
• Hygiena® is a registered trademark of Hygiena.
• Other brand or product names are trademarks of their respective holders.

Reference Number

• The reference number and original Hygiena Diagnostics GmbH article number: is S 400 18.

Change Index

• Version 1, October 2020:
• New document layout and content.
• Version 2, February 2022:
• Rebranding.
• Revision A, September 2023
• New formatting, images, and minor edits
• S 400 18 20-2 -> INS-KIT230187-2-REVA
• Hygiena®
• Camarillo, CA 93012

USA

• diagnostics.support@hygiena.com

Manufactured by Hygiena Diagnostics GmbH

• Hermannswerder 17
• 14473 Potsdam
• Germany
• www.hygiena.com

Documents / Resources

| Hygiena KIT230187 Vibrio Detection LyoKit [pdf] Instruction Manual
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