United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-12 Page 1 of 6

Title: BAX® System Real-time PCR Assay for Escherichia coli O157:H7 in Fresh Produce

Revision: 1 Replaces: 7/1/2010 Effective:09/01/2011

1. Purpose

To provide a standard procedure for detection of Escherichia coli (E. coli) O157:H7 in fresh 

produce using the BAX® system by all laboratories participating in the USDA/AMS 

Microbiological Data Program (MDP). 

2. Scope

This standard operating procedure (SOP) shall be followed by all laboratories conducting 

microbiological studies for MDP, including support laboratories conducting non-routine 

activities. This SOP represents minimum MDP requirements and is presented as a general 

guideline. Each laboratory shall have written procedures that provide specific details 

concerning how the procedure has been implemented in that laboratory.

3. Principle

The BAX® Realtime PCR system is a DNA-based method developed by DuPont Qualicon

for detecting bacterial pathogens in food and environmental samples. The sensitivity and the 

accuracy are a result of the use of polymerase chain reaction (PCR) to amplify DNA 

fragments unique to the target organism. The specific amplification of target genes is

monitored in realtime via the generation of fluorescent signals. 

4. Safety

E. coli O157:H7 is a human pathogen with a low infectious dose. Laboratory personnel 

should utilize Biosafety Level II (BSL-2) practices for microbiological manipulations of 

known and potential pathogens. A BSL-2 laminar flow biosafety cabinet is recommended 

for activities with potential for producing aerosols of pathogens. Material Safety Data Sheets 

(MSDS) should be obtained from manufacturers for media, chemicals and reagents used in 

the analysis and personnel who will handle the materials should know the location of and 

have ready access to the MSDS sheets for reference.

5. Outline of Procedures

Equipment and Materials 7.1

Controls 7.2

Positive Samples 7.3

BAX® Analysis 7.4

Data Analysis 7.5

Reporting 7.6
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-12 Page 2 of 6

Title: BAX® System Real-time PCR Assay for Escherichia coli O157:H7 in Fresh Produce

Revision: 1 Replaces: 7/1/2010 Effective:09/01/2011

6. References

 BAX®System Real-Time PCR Assay – E. coli O157:H7, Part D14203648 DuPont 

Qualicon.

 USDA Microbiological Data Program (MDP) 2010 Multi-Laboratory Method 

Verification Study: Realtime PCR assays for detecting shiga toxin DNA sequences of E. 

coli (STEC) O157:H7 and/or non-O157 serotypes. May 2010.

 BAM Online, Chapter 4a: Diarrheagenic Escherichia coli. Last updated: 08/2009. 

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalytical

ManualBAM/UCM070080 (last accessed 06/2010)

 U.S. Department of Health and Human Services, Public Health Service, Centers for 

Disease Control and Prevention and National Institutes of Health: Biosafety in 

Microbiological and Biomedical Laboratories (BMBL), 5th Edition. 

http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

 SOP MDP-DATA-01 Record Keeping and Results Reporting.

 SOP MDP-LABOP-02 Sample Receipt, Elution, Preenrichment, and DNA Extraction.

 SOP MDP-MTH-06 Escherichia coli O157 Immunomagnetic Separation (IMS) Method 

and Presumptive Confirmation.

 SOP MDP-QA-03 Quality Assurance (QA) Controls.

7. Procedures

7.1 Equipment and Materials

 BAX® Q7 System, DuPont Qualicon

 BAX®System Real-time PCR assay kit for E. coli O157:H7, DuPont Qualicon

 BAX®lysis buffer without protease. Do not add protease to lysis buffer.

 Additional materials needed to perform procedure as listed in BAX®System Real-Time 

PCR Assay for E. coli O157:H7 Kit Insert

7.2 Controls (Specific strains are listed in SOP MDP-QA-03) - Carry all controls through 

this entire procedure, including any necessary cultural confirmation. If any of the controls 

fail to yield a satisfactory result refer to SOP MDP-QA-03.

 Uninoculated Media Control (DNA from uninoculated UPB)
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-12 Page 3 of 6

Title: BAX® System Real-time PCR Assay for Escherichia coli O157:H7 in Fresh Produce

Revision: 1 Replaces: 7/1/2010 Effective:09/01/2011

 No Template Control: Transfer 27 uL of lysis buffer without protease and 3 uL of PCR 

grade water.

 Negative Cultural Control: DNA from E. coli (MDP-017) negative culture control from

SOP MDP-LABOP-02.

 Positive Cultural control: DNA from MDP-004 E. coli O157:H7 positive culture control 

from SOP MDP-LABOP-02.

 Positive Produce Control: DNA from inoculated produce culture control from SOP MDP 

MTH-11 and refer to SOP MDP LABOP-02. 

7.3 Positive Samples - STEC pooled and/or individual positive samples (Rt-PCR positive for 

any one or combinations of stx1, stx2 and uidA genes) from SOP MDP MTH-11. Refer to 

SOP MDP-LABOP-02 for commodity-specific DNA sample source. (Keep DNA 

refrigerated or in cold block until ready to use.)

7.4 BAX® Analysis

7.4.1 Refer to the BAX® System Real-Time PCR Assay for E.coli O157:H7 Kit insert

for equipment set-up and sample loading procedures. Do not perform Kit Insert steps, 4.1 

through 4.7 and at step 5.4, instead do the following: 

7.4.1.1 Hydrate each BAX®reagent pellet by adding 27 μL of BAX®lysis buffer 

(without protease) to the PCR tubes. Do not add DNA sample directly to the BAX® 

reagent pellet without prior addition of lysis buffer (without protease). 

7.4.1.2 Transfer 3μL of undiluted extracted DNA prepared from each UPB enriched 

samples and controls (from SOP MDP-LABOP-02) to the appropriate BAX®PCR 

tubes and seal the tubes. 

Note: To minimize contamination, keep samples and controls separate. PCR tablets 

shall be hydrated and re-sealed within 10 minutes after removing the caps from the 

PCR tubes.

7.4.2 Return to BAX® Real-time O157 Kit insert and complete remaining steps, 6.1 

through 6.3, to begin automated processing of samples. 

7.4.3 For positive BAX® Real-time O157 assay results proceed to SOP MDP-MTH-06,

Escherichia coli O157 Immunomagnetic Separation (IMS) and Cultural Methods. 
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-12 Page 4 of 6

Title: BAX® System Real-time PCR Assay for Escherichia coli O157:H7 in Fresh Produce

Revision: 1 Replaces: 7/1/2010 Effective:09/01/2011

7.4.4 For negative BAX® Real-time O157 assay results, refer to SOP MDPDATA-01 for reporting requirements.

7.4.5 For Indeterminate BAX® Results

7.4.5.1 Remove the refrigerated original pooled and/or individual extracted DNA 

samples and centrifuge the DNA samples for approximately 10 seconds. Reanalyze the centrifuged DNA samples according to section 7.4. of this SOP.

7.4.5.2 If any individual sample shows an indeterminate result, proceed with SOP 

MDP-MTH-06 Escherichia coli O157 Immunomagnetic Separation (IMS) Method

and Presumptive Confirmation for cultural confirmation of individual BAX®

positive samples.

7.5 Data Analysis - Refer to section 7 of the BAX® Realtime PCR Kit insert for results 

interpretation guidance.

7.6 Reporting

7.6.1 A BAX® Real-time PCR O157 assay positive result without cultural confirmation 

and identification is considered a preliminary positive.

7.6.2 Data shall be reported according to SOP MDP-DATA-01.

Disclaimer: Reference to brand names (kits, equipment, media, reagents, etc.) does not 

constitute endorsement by this agency.
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-12 Page 5 of 6

Title: BAX® System Real-time PCR Assay for Escherichia coli O157:H7 in Fresh Produce

Revision: 1 Replaces: 7/1/2010 Effective:09/01/2011

Mary Tijerina 31 August 2011

Revised by: Mary Tijerina Date

Microbiologist, Monitoring Programs Division

8609 Sudley Rd, Ste 206, Manassas, VA 20110

(703) 330-2300 x111

Cynthia Mangione 1 September 2011

Reviewed by: Cynthia Mangione Date

Chairperson, MDP Technical Advisory Group

New York Department of Agriculture & Markets

Food Laboratory, Bldg 7, State Office Campus

1220 Washington Ave, Albany, NY 12235

(518) 457-0906

Shanker Reddy 1 September 2011

Reviewed by: Shanker Reddy Date

Molecular Biologist, Monitoring Programs Division

8609 Sudley Road, Ste 206, Manassas, VA 20110

(703) 330-2300 x135

Diana Haynes 1 September 2011

Approved by: Diana Haynes Date

Deputy Director, Monitoring Programs Division

8609 Sudley Road, Suite 206, Manassas, VA 20110

(703) 330-2300x134

ELECTRONICALLY REPRODUCED SIGNATURES - ORIGINAL SIGNATURE PAGE MAINTAINED BY USDA, AMS, S&T, MONITORING PROGRAMS OFFICE
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-12 Page 6 of 6

Title: BAX® System Real-time PCR Assay for Escherichia coli O157:H7 in Fresh Produce

Revision: 1 Replaces: 7/1/2010 Effective:09/01/2011

Revision 1 August 2011 Monitoring Programs Division

 Updated Outline of Procedures, Section 5.

 Replaced the word “must” with the word “shall” in Note under section 7.4.1.2 

 Deleted Section 7.4.3 and following Note due to no longer using mEC+n, “For positive 

pooled and/or individual BAX® Real-time samples, transfer 25ml from individual UPB 

enriched samples to 225ml mEC+n broth and incubate at 42 ± 2°C for 18-24 hours. Note:

If this step 7.4.3 has been previously completed per SOP MDP-MTH-11, do not repeat.”

 Renumbered SOP sections.

 Added text to (new) Section 7.4.3 to indicate when to refer to SOP MDP-MTH-06.