United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-04 Page 1 of 9

Title: Detection of Salmonella in Fresh Produce by BAX® PCR

Revision: 05 Replaces: 09/25/09 Effective: 07/01/2010

1. Purpose

To provide a standard procedure for detection of Salmonella species 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®PCR system is a DNA-based screening method developed by DuPont Qualicon for

detection of 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.

4. Safety

Laboratory personnel should utilize Biosafety Level II (BSL-2) practices for MDP

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
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-04 Page 2 of 9

Title: Detection of Salmonella in Fresh Produce by BAX® PCR

Revision: 05 Replaces: 09/25/09 Effective: 07/01/2010

BAX® Analysis 7.3

BAX®Positive Samples 7.4

Data Analysis 7.5

Reporting 7.6

6. References

 BAX®System User Guide & Protocol Summary, DuPont Qualicon

 SOP MDP-DATA-01 Record Keeping and Results Reporting

 SOP MDP-LABOP-02, Sample Receipt, Elution, Pre-enrichment, and DNA Extraction

 SOP MDP-MTH-09, Detection of Salmonella using VIDAS® Method

 SOP MDP-MTH-10, Isolation and Identification of Salmonella using Cultural Methods

 SOP MDP-QA-03, Quality Assurance (QA) Controls

7. Procedures

7.1 Equipment and Materials

 BAX® Q7 System, DuPont Qualicon

 BAX®PCR assay kit for Salmonella species, DuPont Qualicon

 Additional materials needed to perform procedure as listed in BAX®System User

Guide & Protocol Summary

7.2 Controls (Specific strains are listed in SOP MDP-QA-03)
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-04 Page 3 of 9

Title: Detection of Salmonella in Fresh Produce by BAX® PCR

Revision: 05 Replaces: 09/25/09 Effective: 07/01/2010

Carry all controls through this entire procedure, including any necessary cultural confirmation.

Refer to SOP MDP LABOP-02 for control setup.If any of the controls fail to yield a satisfactory

result refer to SOP MDP-QA-03.

 Uninoculated Media Control: DNA fromuninoculated Universal Preenrichment Broth (UPB)

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

from SOP MDP-LABOP-02

 Positive Cultural Control: DNA from Salmonella typhimurium (MDP-014) positive culture

control from SOP MDP-LABOP-02

 Positive ProduceControl: DNA from inoculated produce culture control from SOP MDPLABOP-02

 No-template Control: Transfer 45μL of BAX®lysis buffer (without protease) and add 5 uL of

PCR grade water.

7.3 BAX

® Analysis

7.3.1Refer to the BAX® User Manual for BAX®run set-up and sample loading procedures.

7.3.2 For all commodities except alfalfa sprouts, use DNA extracted from the pooled UPB

pre-enriched samples. Keep the DNA under refrigeration or in a cooling block until ready to

use. Refer to SOP MDP-LABOP-02.

7.3.3 For alfalfa sprouts, use DNA extracted from individual UPB pre-enriched samples.

Keep the DNA under refrigeration or in a cooling block until ready to use. Refer to SOP

MDP-LABOP-02.

7.3.4 Transfer enough BAX® PCR tubes for all pooled/non-pooled (sprouts) samples and

controls to the cooling block.

7.3.5 Hydrate each BAX®reagent pellet by adding 45μL of BAX®lysis buffer (without

protease) to the PCR tubes

7.3.6 Transfer 5μL of extracted DNA prepared from each of the UPB-pre-enriched samples

and controls (from SOP MDP-LABOP-02) to the appropriate BAX® PCR tubes. Do not add
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-04 Page 4 of 9

Title: Detection of Salmonella in Fresh Produce by BAX® PCR

Revision: 05 Replaces: 09/25/09 Effective: 07/01/2010

DNA sample directly to the BAX®reagent pellet without prior addition of lysis buffer

(without protease).

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

7.3.7 Start BAX® PCR analysis according to the BAX® User Manual.

7.4 BAX®Positive Samples

7.4.1 When a pooled sample is BAX®positive, extract DNA from individualsamples that

constituted this pool by following SOP MDP-LABOP-02.

7.4.2 Transfer 5μL of extracted DNA from the positive pool and individual samples, along

with appropriate controls, tohydrated BAX® PCR tubes(section 7.3.5) and run the analysis.

7.4.3 Proceed to SOP-MTH-09, Detection of Salmonella using VIDAS® method, for

secondary testing of pooled and/or individual BAX®positive samples. A pooled positive

BAX®result followed by pooled and individual negative VIDAS®results and individual

negativeBAX®results will be recognized as a confirmed negative. If any questions, consult

MPO.

7.4.4 Proceed to SOP MDP-MTH-10, Isolation and Identification of Salmonella using

Cultural Methods for cultural confirmation of individual BAX®positive samples. In order to

increase the likelihood of cultural isolation, laboratories should begin cultural methods as

soon as possible from the individual positive samples as well as from the BAX®positive

pooled culture

7.5 Indeterminate BAX® results

7.5.1 Remove the refrigerated original pooled DNA samples and centrifuge the DNA

samples for approximately 10 seconds. Re-analyze the centrifuged DNA samples

according to section 7.3 of this SOP.

7.5.2 If results are still indeterminate, extract the DNA according to LABOP-02 from

each of the individual refrigerated UPB enriched samples from the indeterminate pooled

sample.

7.5.3 Analyze each individual extracted DNA sample according to section 7.3 of this

SOP.
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-04 Page 5 of 9

Title: Detection of Salmonella in Fresh Produce by BAX® PCR

Revision: 05 Replaces: 09/25/09 Effective: 07/01/2010

7.5.4. If an individual sample shows an indeterminate result, proceed with sections 7.4.3

and 7.4.4 of this SOP.

7.6 Data Analysis Refer to the BAX® User Manual for melting curve interpretation

7.7 Reporting - A BAX®positive result will be reported to MPO as a preliminary positive

result as soon as possible per 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-04 Page 6 of 9

Title: Detection of Salmonella in Fresh Produce by BAX® PCR

Revision: 05 Replaces: 09/25/09 Effective: 07/01/2010

Mary Tijerina 17 June 2010

Revised by: Mary Tijerina Date

Microbiologist, Monitoring Programs Office

8609 Sudley Road, Suite 206

Manassas, VA 20110

(703) 330-2300 x111

Maya Achen 17 June 2010

________________________________________________________________

Reviewed by: Maya Achen Date

Chairperson, MDP Technical Advisory Group

Ohio Department of Agriculture, Consumer Analytical Laboratory, Bldg 3

8995 E Main Street, Reynoldsburg, OH 43068

(614) 728-2790

Kristi McCallum 17 June 2010

____________________________________________________________

Reviewed by: Kristi McCallum Date

Colorado Department of Agriculture

Inspection & Consumer Services Division Laboratory Section

2331 West 31st Avenue

Denver, CO 80211-3859

(303) 477-0014

Shanker Reddy 17 June 2010

Reviewed by: Shanker Reddy , PhD Date

Molecular Biologist, Monitoring Programs Office

8609 Sudley Road, Suite 206, Manassas, VA 20100

(703) 330-2300 x135
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-04 Page 7 of 9

Title: Detection of Salmonella in Fresh Produce by BAX® PCR

Revision: 05 Replaces: 09/25/09 Effective: 07/01/2010

Diana Haynes 17 June 2010

Approved by: Diana Haynes Date

Deputy Director, Monitoring Programs Office

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-04 Page 8 of 9

Title: Detection of Salmonella in Fresh Produce by BAX® PCR

Revision: 05 Replaces: 09/25/09 Effective: 07/01/2010

Revision 05 June 2010 Monitoring Programs Office

 Removed attachment

 Added Safety, Section 4

 Added Outline of Procedures, Section 5

 Updated References, Section 6

 Updated Control list

 Updated section 7.5

Revision 04 September 2009 Monitoring Programs Office

 Updated References, Section

 Revised Section 5.3

 Revised Section 5.4

 Revised Section 5.6

 Added Atch 1, Salmonella Flowchart

Revision 03 March 2009 Monitoring Programs Office

 Deleted “The BAX system is used here as a screening tool” from the Purpose.

 Added Data Analysis to the Outline

 Deleted MDP-MTH-03A from the References and added MDP-MTH-10.

 Added “DuPont Qualicon” to 6.1.1 and 6.1.2

 Deleted “List of” from Controls in the Outline and section 6.2.

 Added “Uninoculated Media Control” and “uninoculated” to 6.2.1.

 Added “Negative Cultural Control” and Escherichia coli (MDP-017)” to 6.2.2.

 Added “Positive Cultural Control” and “Salmonella typhimurium (MDP-014)” to 6.2.3.

 Added “Positive Matrix (produce) Control” and “inoculated” to 6.2.4.

 Deleted the Lysis Buffer control from 6.2.

 Added 6.3.1

 Added “pooled” to 6.3.2. Added SOP reference to 6.3.2.
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No.: MDP-MTH-04 Page 9 of 9

Title: Detection of Salmonella in Fresh Produce by BAX® PCR

Revision: 05 Replaces: 09/25/09 Effective: 07/01/2010

 Added “enough BAX” and “for all pooled/non-pooled (sprouts) samples and controls” to

6.3.3.

 Added “each” to 6.3.5.

 Deleted “aliquot” from 6.3.6 and added “appropriate BAX” to 6.3.5.

 Added 6.3.7 and deleted “Proceed as directed by BAX User Manual”.

 Added 6.4.1 procedure for BAX positive pooled samples.

 Added 6.4.2 for indeterminate BAX results.

 Added 6.5 Data Analysis.

Revision 02 January 2006 Monitoring Programs Office

 Added references to UPB

 Modified BAX®procedure to allow extracted DNA

 Screening procedure now performed on individual samples (rather than 3 pooled samples)

 All cultural procedures now included in SOP MDP-MTH-03A

Revision 01 January 2005 Monitoring Programs Office

 Removed references to outdated control strains

 Removed all duplicative instructions found in BAX® Users Manual

 Added provision to obtain 25 mL wash eluate for positive produce control