BAX® System Real-Time PCR Assay
E. coli O157:H7 Exact
Part KIT2039

KIT CONTENTS

96 PCR tubes with tablets (2 bags 6 x 8 strips)
96 flat optical caps (12 x 8 strips)
1 bottle of protease (400 µL)
2 bottles of lysis buffer (12 mL)

INTENDED USE

Food processors and associated laboratories can use the BAX® System as a quick and reliable method for detecting E. coli O157:H7 in a variety of foods, hemp and cannabis flowers. This real-time PCR assay was designed to report yes/no results  for the presence
of E. coli O157:H7 at concentrations as low as 104 cfu/mL after enrichment. With a processing time of approximately 55 minutes in the BAX® System Q7 instrument, the method returns results with an accuracy that is comparable to culture methods, but with a significantly faster time to result. BAX® Systems are designed for use by qualified lab personnel who follow standard microbiology laboratory practice, including the safe handling and disposal of potentially pathogenic materials. The laboratory must comply with good laboratory practice (see ISO 7218 standard).
Field of use : Data obtained from the BAX® System should not be used for human diagnostic or human treatment purposes. Equipment is not approved by the United States Food and Drug Administration or any other U.S or non-U.S. regulatory agency for use in human diagnostics or treatment. The BAX® System should not be used as the sole basis for assessing the safety of products for release to consumers. The information generated is only to be used in conjunction with the user’s regular quality assurance program. Not approved for clinical diagnosis. Use for research and development, quality assurance and quality control under supervision of technically qualified persons.

PRINCIPLE OF THE METHOD
See the BAX® System User Guide for an overview of how the BAX® System method uses automated, real-time Polymerase Chain Reaction (PCR) technology.
MATERIALS
BAX® System Real-Time PCR Assay for E. coli O157:H7 Exact (Part KIT2039)
BAX® System start-up package (equipment and supplies for up to 192 tests)

• BAX® System Q7 cycler/detector and computer workstation
• Heating blocks with inserts* capable of maintaining 37±2ºC and 95±3ºC
• Cooling blocks with inserts*
• PCR tube holder
• Capping/decapping tools
• Adjustable mechanical pipettes (5-50 µL; 20-200 µL)
• Repeating pipette
• Multi-channel pipette (8 channels ­ 5-50 µL)
• Cluster tubes with caps and racks
• Pipette tips with barriers
• Powder-free nitrile gloves

*The Automated Thermal Block (Catalog No. MCH2023) may be used in place of heating and cooling blocks.
Stomacher with bags. Incubator capable of maintaining directed enrichment temperatures within ±2°C.
Note: Health Canada, AOAC, and AFNOR Certification standards require an incubator capable of maintaining ±1°C.
Enrichment media (See BAX® System User Guide for details)

• BAX® System MP Media ­ Catalog No. MED2003 (2.5 kg)

Note: StatMediaTM soluble packets may also be used to prepare BAX® System MP media. See instructions on packet or in User Guide.

STORAGE AND SHELF LIFE

• Reagents and PCR tubes with tablets should be kept refrigerated at 2­8°C. Do not freeze.
• Reagents should be used by the expiration date stamped on the individual labels.
• After protease has been added to the lysis buffer, shelf life of the solution is 2 weeks when stored at 2-8°C.
• If storing PCR tubes with tablets in an open kit for more than 3 weeks, seal the Mylar bag of PCR tubes into a larger bag with desiccant or store at 4ºC in a desiccation unit, if possible.

SAFETY PRECAUTIONS

The BAX® System method includes sample enrichment procedures that nourish the growth of potential pathogens to detectable levels. Because pathogens can cause human illness, appropriate safety precautions must be taken and personal protective equipment worn when handling samples, media, reagents, glassware and other supplies and equipment that could be contaminated with potentially pathogenic bacteria.
Reagents used with the BAX® System assays should pose no hazards when used as directed. Before using this product, please review the Safety Data Sheets (SDS) included with your BAX® System purchase and also available at www.hygiena.com. Refer to your site practices for safe handling of materials at extreme temperatures.

SOFTWARE REQUIREMENTS

Before using this assay for the first time, install the most current version of the BAX® System software, then run a calibration report to check that “Real Time E. coli O157:H7 Exact” appears in the list of calibration files. See “Troubleshooting Calibration” in the BAX® System User Guide for details. If the report list does not contain “Real Time E. coli O157:H7 Exact”, you must recalibrate the Q7 instrument to load the required dyes. Be sure to allow enough time to complete the calibration (about 1.5 to 2 hours) before starting the assay. For instructions and tips on calibrating the instrument, see the BAX® System User Guide.

ENRICHMENT PROTOCOL

1. Prepare Enrichment Broth
   Dissolve 22.5 g BAX® System MP media in 1 L distilled water and mix. Do not boil. Adjust pH to a final value of 7.2±0.2 at 25°C, then autoclave at 121°C for 15 minutes.

2. Collect and Enrich Samples

• Raw ground beef: For 25 g: Homogenize 25 g sample with 225 mL pre-warmed (42°C) BAX® System MP media or mTSB (Modified Tryptic Soy Broth).
• Incubate at 42°C for 8-24 hours. For 375 g: Homogenize 375 g sample with 1.5 L pre-warmed (45°C) BAX® System MP media or mTSB. Incubate at 42°C for 8-24 hours.
• Beef trim: MP Media: Mix 375 g sample by hand with 1.5 L pre-warmed (45°C) BAX® System MP media. Incubate at 42°C for 8-24 hours.
• Beef trim: mTSB: Mix 375 g sample by hand with 1.5 L pre-warmed (45°C) mTSB. Incubate at 42°C for 10-24 hours.
• Spinach and lettuce: 375g: Combine 375 g sample with 1.5 L pre-warmed (42°C) BAX® System MP media. Incubate at 42°C for 6-24 hours.
• Raw Milk: Swirl 25 mL of sample with 225 mL prewarmed (41.5°C) double strength BPW (Buffered Peptone Water). Incubate at 41.5°C for 12-24 hours.
• Dried cannabis and hemp flower: Combine 10 g flower with 90 mL prewarmed (37-42°C) BPW. Hand massage for 1 minute and incubate at 42°C for 22-26 hours.
• Sampling Cloths such as MicroTally® (375 g Beef Trim): Combine one sampling cloth with 200 mL pre-warmed (42°C) MP media or mTSB + caa (modified TSB with casamino acids) media. Stomach for 1 minute and incubate at 42 ± 1°C for 8-24 hours.

TEST PROTOCOL

3. Prepare Equipment
3.1 Turn on the heating blocks to 37ºC and 95ºC.
3.2 Make sure cooling blocks are chilled to 2-8ºC.
*If using the Automated Thermal Block, follow the instructions in the Thermal Block User Guide for running the Gram Negative program.
3.3 Power on the Q7 instrument and launch the BAX® System application.
3.4 Create a rack file (see User Guide for details).
4. Perform Lysis
4.1 Break cluster tubes apart.
4.2 Label and arrange cluster tubes in rack according to the rack file.
4.3 Prepare lysis reagent by adding 150 µL protease to one
12 mL bottle of lysis buffer.
4.4 Transfer 200 µL lysis reagent to each cluster tube.
4.5 Transfer 20 µL enriched sample to the corresponding cluster tube.
4.6 Heat at 37°C for 20 minutes.
4.7 Heat at 95°C for 10 minutes.
4.8 Cool at 2-8°C for at least 5 minutes.
5. Hydrate PCR Tablets
5.1 Initialize the instrument by selecting RUN FULL PROCESS from the OPERATION menu.
5.2 Place a PCR tube rack onto a chilled (2-8°C) PCR cooling block.
5.3 Arrange strips of PCR tubes according to your rack file.
5.4 Remove the caps from the first strip of tubes with the decapping tool. 5.5 Transfer 30 µL lysate (from step 4.8) into PCR tubes, then seal with flat optical caps.
5.6 Repeat with remaining strips of PCR tubes until all PCR tablets have been hydrated.
Note: PCR tablets must be hydrated and re-sealed immediately after removing the caps from the PCR tubes.
5.7 After the completion of hydration of all PCR tablets, let PCR tubes sit in the cooling block for 10-30 minutes before loading into the BAX® System instrument. Note: Do not let PCR tubes sit for more than 30 minutes.
6. Amplify and Detect
6.1 At the “Ready for Rack Load” prompt, click the NEXT button and open the instrument drawer.
6.2 Place the rack of PCR tubes over the wells in the drawer and check that the tubes are seated correctly.
6.3 Close the drawer and click the NEXT button to begin automated processing.
7. Review Results
Qualitative results are displayed as a grid of color-cued icons in the top half of the screen:

*Refer to the troubleshooting section in the User Guide for assistance.

CONFIRMATION

Method Approved by AOAC.
If desired, BAX® method enriched samples can be confirmed with the reference culture method appropriate for the sample type, such as:

• U.S. FDA Bacteriological Analytical Manual (BAM)
• USDA FSIS Microbiology Laboratory Guidebook (MLG)
• International Organization for Standardization (ISO)
• AOAC SMPR 2020.002

All samples identified as positive by the BAX® System method must be confirmed in one of the following ways:

• Direct plating- Streak 50 µL of enrichment onto CT-SMAC and Rainbow agar or mRBA (Modified Rainbow agar). Incubate plates for 18-24 hours at 37ºC. Test 1-5 typical colonies from at least one selective agar plate by:
  • Latex agglutination and BAX® System RealTime Assay for E. coli O157:H7 Exact.
• Immuno-concentration (IMS) – Perform IMS and streak 50 µL of enrichment onto CT-SMAC and Rainbow agar or mRBA. Incubate plates for 18-24 hours at 37ºC. Test 1-5 typical colonies from at least one selective agar plate by:
  • Latex agglutination and BAX® System RealTime Assay for E. coli O157:H7 Exact.

If confirming a colony with the BAX® method, follow these steps:

1. Examine selective agar plates for suspect colonies and identify those that require confirmation.

2. Prepare lysis tubes according to the screening protocol and fill out your sample tracking sheet.

3. Add 1 mL of sterile diluent to sterile tubes, and label them to correspond with your lysis tubes.
   Note: If confirming samples with the BAX® System Real-Time E. coli O157:H7 Exact, use MP media or mTSB as the sterile diluent.
   Note: If your BAX® System results are positive, you can then use these tubes to streak plates for isolation and further identification of the target organism.

4. Touch a plastic disposable needle to the agar plate to pick a suspect colony.
   Note: Avoid picking larger colonies, as adding too much sample DNA to the dilution tube may affect results.

5. Dip the needle into the corresponding dilution tube and swirl the needle to release the colony. Remove the needle, then cap the tube and mix.

6. Uncap the dilution tube, transfer 20 L of the cell suspension into the corresponding lysis tube containing 200 L lysis reagent, and mix.

7. Repeat steps 4-6 for each of your suspect colonies, using a new needle for each pick.

8. Continue with the BAX® System testing protocol from “Perform Lysis.”

DISPOSAL

Decontaminate materials and dispose of biohazardous waste per your site practices and as required by federal, state and local regulations.

VALIDATION

The BAX® System Real-Time PCR Assay for E. coli O157:H7 EXACT has been certified by the AOAC Research Institute as Performance Tested MethodSM

102003. This test kit’s performance was reviewed by AOAC-RI and was found to

perform to the manufacturer’s specifications. Validation studies for foods, hemp and cannabis flowers demonstrated BAX® System sensitivity and specificity equal to or better than the reference culture-based methods.

TECHNICAL ASSISTANCE

For questions or comments, please contact your local distributor. You can also call 1-800-863-6842 in the U.S., 1302-695-5300 outside the U.S., or email diagnostics.support@hygiena.com.

LIMITATION OF WARRANTY AND LIABILITY

NOTICE: READ THIS LIMITATION OF WARRANTY AND LIABILITY BEFORE USING THE BAX® SYSTEM EQUIPMENT, ASSAYS, AND/OR MEDIA (“BAX® SYSTEM”). If the terms are not acceptable, notify Hygiena immediately and arrangements will be made for return of the unused Equipment, assays, and/or media to Hygiena and for the refund of the purchase price, less shipping costs. USE OF BAX® SYSTEM EQUIPMENT, ASSAYS AND/OR MEDIA CONSTITUTES AN ACCEPTANCE OF ALL TERMS AND CONDITIONS OF THIS LIMITATION OF WARRANTY AND LIABILITY. Any additional or different terms in Buyer’s purchase form(s) are material alterations and hereby rejected.

1. BAX® System Equipment should only be used with BAX® System assays.
2. When used with BAX® System assays, BAX® System Equipment is warranted be free of defects in materials, workmanship and design that may appear under normal and proper use within twelve (12) months from the installation date to the first end user. BAX® System assays are warranted to conform to the assay description under the conditions of use specified in the user documentation to the expiration date stamped on the label. BAX® System media is warranted to meet standard specifications in effect on the date of shipment. Hygiena MAKES NO OTHER WARRANTY, EITHER EXPRESS OR IMPLIED, INCLUDING, WITHOUT LIMITATION, ANY WARRANTY AGAINST INFRINGEMENT, ANY WARRANTY OF MERCHANTABILITY OR OF FITNESS FOR A PARTICULAR PURPOSE OR THOSE ARISING BY LAW, STATUTE, USAGE OF TRADE, OR COURSE OF DEALING. User assumes all risk and liability resulting from use of the BAX® System Equipment, assays and media, whether used singly or in combination with other products.
3. BAX® Software: Hygiena warrants that for a period of 60 days from the date of first date of use by the Customer/end user, BAX® software media will be free from defect in materials and workmanship and that the BAX® software will substantially perform in accordance with the accompanying BAX® software documentation. EXCEPT FOR THE EXPRESS WARRANTY ABOVE, HYGIENA MAKES NO OTHER WARRANTY, EITHER EXPRESS OR IMPLIED, INCLUDING, WITHOUT LIMITATION, ANY WARRANTY AGAINST INFRINGEMENT, ANY WARRANTY OF MERCHANTABILITY OR OF FITNESS FOR A PARTICULAR PURPOSE OR THOSE ARISING BY LAW, STATUTE, USAGE OF TRADE, OR COURSE OF DEALING. User assumes all risk and liability resulting from use of the BAX® software, whether used singly or in combination with other products.
4. The accuracy of the BAX® System can be affected by factors over which Hygiena has no control, including, without limitation, the use of the Equipment, assays and/or media in a manner that is contrary to the conditions of use, the procedures or the instructions specified by Hygiena. Because of the large number of factors over which Hygiena has no control, Hygiena makes no promise or guarantee of the accuracy of, or results obtained from the use of the BAX® System. In particular, Hygiena disclaims any warranty or liability and assumes no responsibility whatever for the failure of the BAX® System due, in whole or in part, to user’s failure to: (a) properly maintain Equipment, (b) maintain specified operating or storage conditions, (c) follow the specified instructions, or (d) use the proper microbiological techniques consistent with the standard of care accepted in the industry for the proper collection, storage, handling and preparation of the sample.
5. Externally caused failures, such as improper sample preparation, improper storage or loading of reagents, electrical outages, or out-of-specification environmental conditions are not covered under this warranty. Equipment failures caused by spills, abuse, misuse, negligence, or improper operation are not covered by this warranty. Modifications, service or repairs by parties other than Hygiena-authorized providers are not covered by this warranty and, in fact, void this warranty. Circumstances beyond the reasonable control of Hygiena, including fire, explosions, accidents, flood, labor trouble or shortage, war, act of or authorized by any government, inability to obtain suitable material, Equipment, fuel, power or transportation, or acts of God are not covered under this warranty.
6. The BAX® System is designed to test only for the presence of the target organisms specified in the particular assay. The BAX® System has been tested against many, but not all, strains of the target within the sample types specified in the user documentation. Hygiena, therefore, cannot and does not make any representation or warranty that the BAX® System is capable of detecting every organism in the target genus, serotype, or species in any sample source. Accordingly, the BAX® System should not be used as the sole test for the release of user’s products, nor should it be used as the sole basis for determining the safety of user’s products.
7. CUSTOMER/USER ASSUMES ALL RISKS IN USING THE BAX® SYSTEM AND HYGIENA OR ITS AFFILIATES, DISTRIBUTORS, ITS LICENSORS OR REPRESENTATIVES SHALL HAVE NO LIABILITY TO CUSTOMER/USER OR TO ANY OTHER  PERSON OR ENTITY FOR ANY INDIRECT, INCIDENTAL, SPECIAL, PUNITIVE, EXEMPLARY OR CONSEQUENTIAL DAMAGES WHATSOEVER, INCLUDING, BUT NOT LIMITED TO, LOSS OF REVENUE OR PROFIT, LOST OR DAMAGED DATA OR OTHER COMMERCIAL OR ECONOMIC LOSS EVEN IF CAUSED BY THE NEGLIGENCE OF HYGIENA OR ITS REPRESENTATIVES AND/OR IF HYGIENA HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES, AND/OR IF THEY ARE FORESEEABLE.
8. THE SOLE AND EXCLUSIVE REMEDY OF CUSTOMER/USER, AND THE SOLE AND EXCLUSIVE LIABILITY OF HYGIENA, ITS AFFILIATES, DISTRIBUTORS, LICENSORS OR REPRESENTATIVES FOR ANY AND ALL CLAIMS, INCLUDING BREACH OF WARRANTY, TORT, CONTRACT, STRICT LIABILITY, NEGLIGENCE OR OTHERWISE SHALL BE LIMITED TO THE FOLLOWING: (a) Should Equipment fail to conform with the Paragraph 2 warranty, Hygiena shall, at its option: repair or replace the non-conforming Equipment with new or refurbished (repaired or rebuilt) functionally equivalent Equipment or refund the purchase price; (b) Should BAX® Software fail to conform with the Paragraph 3 warranty, Hygiena will replace it free of charge; (c) For all other claims, Hygiena may, at its option, refund the purchase price or replace the Equipment, assays or media; (d) In all cases, user is responsible for the repackaging and return of nonconforming Equipment, along with the reinstallation of new or refurbished Equipment; and (e) Equipment, assays or media shall not be returned without prior written permission from Hygiena, and then only in the manner prescribed by Hygiena. The maximum liability of Hygiena, its affiliates, distributors and licensors, and whether or not based on negligence, shall not exceed in the aggregate the amount equal to: (a) the purchase price of the BAX® System, assay or media for which damages are claimed, or (b) in the case of BAX® Software, the amount paid for the software (if licensed separately) or twenty-five thousand dollars ($25,000.00USD). Customer/user shall notify Hygiena of any claim within thirty (30) days thereof and shall commence any action against Hygiena within one (1) year of the cause of action or otherwise be barred from any remedy. Hygiena shall not be responsible for cost, loss or liability that arise from customer/user’s operation of its business, and customer/user agrees to indemnify, defend and hold Hygiena and its representatives harmless from such cost, loss or liability.

BAX® System Q7 instruments are sold under licensing arrangement with Applied Biosystems for food testing. Use for research and development, quality assurance and quality control testing under supervision of technically qualified persons.
Not approved for clinical use. Please read the limitation of warranty and liability before use. Some dyes in this product are sold under licensing agreement with Biosearch Technologies, Inc. for R&D use. Note: MicroTally® is a registered trademark of Fremonta. INS2033 Rev. C Effective date: 5/19/2023

References

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