United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 1 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

1. Purpose

To provide a standard procedure for the detection of Shiga-toxin producing Escherichia coli

(STEC), serotype O157 and non-O157 in commodities analyzed for 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

STECs are detected by a multiplex real-time PCR assay specific for the stx-1, stx-2 genes and 

the +93 uidA mutation unique to E. coli O157:H7. This assay screens for the presence of 

STECs, including E. coli O157:H7 in foods using the Cepheid Smart Cycler II platform.

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

Media and Reagents 7.2

List of Controls 7.3

Template and Master Mix Preparation 7.4

Real-time PCR Analysis 7.5
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 2 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

Interpretation of Results 7.6

Isolation and Identification of non-O157 STEC 7.7

Media Composition 7.8 

Reporting 7.9

6. References

 SOP No: FERN-MIC.0003.00E, Procedures for the Detection of Shiga-toxin 

Escherichia coli (STEC), serotype O157 and non-O157 in Food – Detection by Real-time 

PCR using Cepheid Smart Cycler II. Effective: 08-13-07

 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)

 BAM Online, Media Preparation, m192A

 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.

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

http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm. U.S. Department of Health and 

Human Services, Public Health Service, Centers for Disease Control and Prevention and 

National Institutes of Health: 

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

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

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

 VITEK® Users Manual, bioMérieux
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 3 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

7. Specific Procedures 

7.1 Equipment and Materials 

 Smart Cycler II system, capable of performing cycling parameters described in this 

SOP and simultaneous real-time detection for FAM, TET, Texas Red and Cy5 

fluorophores, Cepheid.

 Smart Cycler reaction tubes, 25 μl minimum capacity, Cepheid

 Smart Cycler reaction tube cooling block, Cepheid

 Smart Cycler reaction tube centrifuge

 VITEK®System or VITEK®2 Compact System, bioMérieux

 VITEK® GNI+ Card or GN cards, bioMérieux

 Vortex mixer

 Appropriate micropipettors 

 Pipet tips, sterile, filter, appropriate sizes 

 Microcentrifuge tubes, 1.5 ml

 Incubators: 35 ± 2oC, 42 ± 2oC and 44 ± 2oC

7.2 Media and Reagents

 TP Broth

 Modified Buffered Peptone Water plus pyruvate (mBPWp) with ACV

 Chromagenic agar plates (Example: DRG Chromagenic agar, etc.)

 L-EMB Agar

 MacConkey Agar

 LST broth

 PCR grade water, nuclease free

 OmniMix® HS PCR master mix reagent beads, or equivalent, containing 3 units 

TaKaRa hot start Taq polymerase, 200 μM dNTPs, 4 mM MgCl2, and 25 mM 

HEPES pH 8.0 ± 0.1.

 10 μM Working Solution of each primer and probe listed in Table 1 or use CSR bead 

available from BioGX. The CSR bead contains all of the primers and probes listed in 

Table 1. (http://www.biogx.com/BioGX_Home/BioGX_Products.html). 

o Stock and Working probe and primer solutions can be prepared using 

commercially synthesized primers with basic desalt purification 

(Fisher/Genosys or equivalent) by rehydrating with sterile distilled water 
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 4 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

to appropriate concentrations. Store frozen until use.

o Probes should be purchased as RP HPLC purified and labeled as follows:

 Stx-1 probe is 5 ́ Texas Red and 3 ́BHQ2 labeled (Integrated DNA 

Technologies Inc., or equivalent).

 Stx-2 probe is 5 ́ FAM and 3 ́ BHQ1 labeled (Integrated DNA 

Technologies Inc., or equivalent).

 uidA probe is 5 ́ TET and 3 ́ MGB-NFQ labeled, Applied 

Biosystems, or equivalent

 Working solutions of probes should be aliquoted into small 

portions and kept frozen until use.

Table 1: STEC Primer/Probe Sequences

Primer/Probe Name Sequence (5 ́ → 3 ́)

Stx1F934 GTGGCATTAATACTGAATTGTCATCA

Stx1R1042 GCGTAATCCCACGGACTCTTC

Stx2F1218 GATGTTTATGGCGGTTTTATTTGC

Stx2R1300 TGGAAAACTCAATTTTACCTTTAGCA

uidAF241 CAGTCTGGATCGCGAAAACTG

uidAR383 ACCAGACGTTGCCCACATAATT

UidAP266 TET-ATTGAGCAGCGTTGG-MGB/NFQ

Stx1P990 Texas Red – TGATGAGTTTCCTTCTATGTGTCCGGCAGAT – BHQ2

Stx2P1249 6FAM-TCTGTTAATGCAATGGCGGCGGATT-BHQ1

Internal Control55F No sequence available

Contained in STEC CSR bead (BioGX)

Internal Control186R No sequence available

Contained in STEC CSR bead (BioGX)

Internal Control Cy5 No sequence available

Contained in STEC CSR bead (BioGX)
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 5 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

7.3 List of Controls (Specific strains are listed in SOP MDP-QA-03)

7.3.1 Carry all cultural controls from all screening methods previously completed through 

this entire procedure. 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.

 No-template Control: Transfer 20 μl of Master Mix and 5 μl of PCR grade water.

 Negative Culture Control: DNA from MDP-017

 Positive Cultural Control: DNA from MDP-004

 Positive Produce Control: DNA from inoculated produce culture control (MDP-004) 

from SOP MDP LABOP-02

 Media Control for Cultural Confirmation

 Positive DNA Control: MDP-019: grow culture and extract DNA prior to assay 

setup

7.4 Template and Master Mix Preparation

7.4.1 Use extracted DNA from overnight UPB enriched samples from SOP MDPLABOP-02.

7.4.2 Prepare master mix containing all of the components, except template, and in 

sufficient volume to run all samples and controls.

7.4.2.1 When using the OmniMixTM HS reagent beads, they contain reagents for 

two 25μl reactions, therefore they need to be rehydrated accordingly. The CSR 

beads contain all the primer, probe and internal control reagents for performing 

four 25μl PCR amplifications. Two OmniMixTM HS beads per CSR bead is 

required to provide the necessary reagents for four PCR amplifications.

7.4.2.2 To prepare master mix, determine the number of reactions needed. Add 

PCR grade water equal to the number of reactions (n) times 20μl (volume of 

master mix added to each reaction tube). Add the appropriate number of OmniMix 

HS beads followed by the appropriate number of CSR beads. Briefly mix the 

master mix. Store the prepared master mix in the dark at 2-8

oC prior to use. 

Prepared master mix can be stored not more than 48 hours. (Example: For 16 
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 6 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

PCR reactions, add 320μl PCR grade water to a DNA/RNA free sterile microfuge 

tube. Add 8 OmniMix HS beads to the tube. Add 4 CSR beads to the tube.)

The preparation of the Master Mix should be performed in a clean hood. Add 

the PCR grade water to a sterile microcentrifuge tube, add all of the beads, and 

let it sit in a cold block until they fully dissolve. 

7.4.2.3 Aliquot 20μL of Master Mix to each of the Smart Cycler reaction tubes 

using the Smart Cycler cold block. Then add 5uL of undiluted DNA sample 

(template) or control template for a final volume of 25μl in appropriate reaction 

tube. 

7.4.2.4 Close the reaction tubes and briefly centrifuge using the Smart Cycler 

centrifuge to bring all liquid to the bottom of the reaction tube. Avoid touching the 

bottom of the Smart Cycler tubes as fingerprints and debris could affect results.

7.4.2.5 Keep all thawed reagents and reagents on ice. Protect CSR beads from 

light.

7.4.2.6 Load the reaction tubes in to the Smart Cycler block and analyze 

according to pre-set protocol specified in step 7.5 of this SOP.

7.5 Real-time PCR Analysis

7.5.1 Create run on SmartCycler II

7.5.2 Give each run a unique run name.

7.5.3 Select Dye set FTTC25

7.5.4 Create and save the following protocol and Select sites used in run. The parameters 

of this cycle are as follows:

7.5.4.1 A 2-step PCR protocol is used with initial activation hold of 60 sec at 
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 7 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

95°C, followed by 40 cycles of 10 sec at 94°C, (optics off) then 40 sec at 

63°C, (optics on).

7.5.4.2 Set analysis settings on SmartCycler® II instrument, for FAM, TET and 

Texas Red channels, and if using CSR beads, include the Cy5 channel. 

Manual Threshold Fluorescence Units for the Cy5 IC is Lot dependent. If 

no Ct is present for the IC in the negative control at default 15.0 fsu, refer 

to Lot specific recommendations.

(1) Usage: Assay

(2) Curve Analysis: Primary

(3) Threshold Setting: Manual

(4) Manual Threshold Fluorescence Units: 15.0

(5) Auto Min Cycle: 5

(6) Auto Max Cycle: 10

(7) Valid Min. Cycle: 3

(8) Valid Max. Cycle: 60

(9) Background subtraction: ON 

(10) Boxcar Avg. Cycles: 0

(11) Background Min. Cycle: 5

(12) Background Max. Cycle: 40 

 

7.5.4.3 Ensure the Manual Threshold Fluorescence Units have been changed to 

15.0. Update analysis settings if they are changed before recording 

results.

7.5.5 Start Run.

7.5.6 Primary fluorescence curves that cross the threshold will be recorded as POS and 

the cycle when the sample crossed the threshold will be recorded in the Results 

Table view (Figure 1A).
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 8 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

7.5.7 Results can be viewed graphically (Figure 1B): A report can be generated or a 

screen capture of the results table view can be used to record data. Table 3 shows 

spectral characteristics of the dyes employed in the assay as well as the available 

optical channels on the Smart Cycler II system. Fluorescence in each channel is 

recorded separately on the instrument

Table 2: Spectral properties of dyes and available channels on the Smart Cycler II

Fluorescent Dye

(nm) Dye

Absorption Max.

(nm)

Emission Max.

FAM 494 518

TET 522 538

TxRd 598 615.5

Cy5 649 670

Smart Cycler II Excitation (nm) Emission (nm) Dye used in

Assay

Ch. 1 450-495 510-527 FAM

Ch. 2 500-550 565-590 TET

Ch. 3 565-590 606-650 TxRd

Ch. 4 630-650 670-750 Cy5

Stx2 presence is recorded in FAM channel (Channel 1)

uidA E. coli O157:H7 clonal type is recorded in TET channel (Channel 2) 

stx1 presence recorded in TxRd channel (Channel 3)

IC (Internal Control) presence recorded in Cy5 channel (Channel 4)
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 9 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

Figure 1A: Results Table View

Figure 1A - Results table view. For each channel the final result, positive or negative, is reported in the 

first column and the Ct at which the positive values crossed the threshold is reported in the second 

column. The FAM, TET, TxRd and Cy5 channels correlate to the stx2, E.coliO157:H7, uidA clonal 

group, stx1 and IC targets respectively.

Figure 1B: Results Graphical View

Figure 1B is a graphical view for the four channels for isolate E.coli O157:H7 ATCC 43895.
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 10 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

7.6 Interpretation of Results

7.6.1 A preliminary positive result for STEC is indicated by DNA detected for a 

respective gene target (stx-1, stx-2, uidA) if indicated as “POS”. 

7.6.1.1 If a pooled sample is positive for any one of the targets, extract DNA from 

individual samples as per SOP MDP-LABOP-02.

7.6.1.2 Setup PCR reactions for the previously extracted pooled and the individual 

samples as per sections 7.2 through 7.5 of this SOP. Also, run same samples 

following SOP MDP MTH-12.

7.6.1.3 Samples that show positive stx-1 or stx-2 (and are negative for MDP 

MTH-12) are considered suspect positive for non-O157 STECs and shall be 

culturally confirmed as per section 7.7 in this SOP. 

7.6.1.4 Samples that show positive uidA results (regardless of stx-1 or stx-2 

results) by SOP MDP MTH-11 and/or positive results by SOP MDP-MTH-12 are 

considered presumptive positive for E. coli O157:H7 and shall be confirmed 

according to SOP MDP MTH-06.

7.6.2 A negative result for STEC is indicated when no DNA is detected for a respective 

gene target (stx-1, stx-2, uidA) if indicated as “NEG” and no cycle threshold value is 

present. 

7.7 Isolation and Identification of non-O157 STEC

Due to the complex nature of isolation, laboratories are encouraged to use best professional 

judgment and experience to obtain a pure culture of the target organism. Isolation and 

identification can be carried out from the pooled sample and the individual sample that tested 

positive in 7.6.1.3
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 11 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

7.7.1 For each STEC positive pooled sample (except for alfalfa sprouts), transfer 10 mL 

of pooled UPB enriched sample to one 90 mL aliquot of mBPWp (with ACV added 

immediately)and incubate at 42 ± 2oC for 18-24 hours. In addition, transfer 10 mL of 

pooled UPB enriched sample to 90 mL TP broth and incubate at 44 ± 2oC for 18-24 

hours.

Note: 10 mL of the pooled sample is left over and if required can be used for additional 

analyses (ex: IMS of the pooled UPB if sample is positive on MDP MTH-12. 

7.7.2 In addition, transfer 25 mL of the positive individual UPB enriched samples into 

225 mL of mBPWp (with ACV added immediately) and incubate at 42 ± 2oC for 18-24 

hours. Also, transfer 25 mL of each of the positive individual UPB enriched samples into 

225 mL of TP broth and incubate at 44 ± 2oC for 18-24 hours. For sprouts, transfer 25 

mL of the individual positive sample accordingly. 

7.7.3 Following incubation, streak duplicate sets of plates of chromogenic agar, L-EMB, 

and MacConkey plates. Incubate one set of plates overnight at 35 ± 2oC and incubate the 

other set of plates overnight at 42 ± 2oC.

Note: Use professional judgment and experience in deciding the number of plates needed 

for picking minimum number of 20 isolated colonies

7.7.4 Examine plates and pick at least 20 typical isolated colonies or swipes from any 

of the selective agar plates (chromogenic, L-EMB, and MacConkey) to appropriate media 

(e.g., LST, BHI, etc.). It is advised to pool multiples of isolates and screen on Rt-PCR to 

determine what isolate(s) that are positive (e.g., pick 20 colonies and pool into 2 groups; 

example: if you have 20 LST samples, split into two groups containing 10 each) and 

perform DNA extraction according to SOP MDP-LABOP-02 and perform Rt-PCR. 

Document procedures used accordingly.

Note: Use professional judgment and experience in deciding the maximum number of 

colonies required for screening which depends on the extent of contamination, the 

background microflora level, type of commodity and post harvest handling.
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 12 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

7.7.5 Identify the individual SmartCycler® Rt-PCR positive culture. Streak the culture to 

selective agar plates for isolation. Incubate overnight at 35 ± 2oC.

7.7.6 Examine plates and if selective agar plates contain typical colonies, streak the 

typical colony onto BA plate and incubate overnight at 35 ± 2oC. Perform VITEK® using 

growth off BA or other appropriate plating media. (If VITEK®identifies the organism as 

a possible E. coli O157:H7, further identification based on cultural tests and serotype is 

required as per SOP-MDP-MTH-06. MPO shall be notified per SOP MDP-DATA-01.) 

Pick 3-5 individual typical colonies to LST or non-specific rich broth. 

7.7.7 Repeat Rt-PCR on 3 isolates. Choose one isolate that is identified as E. coli and 

possesses toxin gene(s) for archiving and shipping according to SOP MDP-SHIP-03. 

7.8 Tryptone Phosphate (TP) Broth - Autoclave for 15 minutes at 121oC. Final pH should be 

7.0 ± 0.2. 

Typical Colony Characteristics of pathogenic E. coli Other Organisms of Interest

Medium Colony Characteristics

MacConkey Red to pink

L-EMB Blue-black and green w/metallic 

sheen

Chromogenic agar refer to manufacturer’s user 

guide

refer to manufacturer’s user 

guide

Tryptone 20.0 g

K2HPO4 2.0 g

KH2PO4 2.0 g

NaCl 5.0 g

Tween 80 1.5 mL

Deionized Water 1.0 L
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 13 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

7.9 Reporting - A “preliminary STEC” is defined as an isolated organism that is shown by RtPCR to possess the DNA sequences associated with the stx1 and/or stx2 genes and to NOT 

possess the E. coli O157:H7 DNA sequence (secondary analysis per MDP-MTH-12). The 

isolate(s) slants are to be shipped to the repository (per MDP-SHIP-03 SOP) for archival and/or 

additional testing. If additional testing is performed by an outside agency, upon receipt of 

PFGE/serological results, MPO will provide test data to repository and originating laboratory. 

Report results 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

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Microbiological Data Program

SOP No: MDP-MTH-11 Page 14 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

Rt-PCR positive UPB cultures (pooled and/or individual)

If selective plates have typical 

colonies, confirm by VITEKusing BA 

growth

Select one Rt-PCR confirmed isolate and 

prepare 3 slants for shipping per MDP SOP 

SHIP-03

Perform BAX® System O157 Rt-PCR Assay on 

positive pooled and/or positive individual 

samples and streak to selective media 

Streak 3 (chromogenic agar, MacConkey, L-EMB) and 

incubate at 35 & 42oC

If VITEK® positive, pick 3 

typical colonies to LST and 

then repeat Rt-PCR

For positive BAX® System 

O157 Rt-PCR samples 

proceed to SOP MDP MTH-06

Flowchart:

STEC Isolation and Identification

Transfer to mBPWp and TP broths as 

described and incubate at appropriate 

temperatures
United States Department of Agriculture

Agricultural Marketing Service, Science & Technology

Microbiological Data Program

SOP No: MDP-MTH-11 Page 15 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

Mary Tijerina 7 July 2011

________________________________________________________________

Revised by: Mary Tijerina Date

Microbiologist, Monitoring Programs Division

8609 Sudley Road, Suite 206, Manassas, VA 20110

(703) 330-2300 x111

Maya Achen 6 July 2011

________________________________________________________________

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

Shanker Reddy 6 July 2011

Reviewed by: Shanker Reddy, Ph.D. Date

Molecular Biologist, Monitoring Programs Division

8609 Sudley Road, Suite 206, Manassas, VA 20110

(703) 330-2300 x135

Diana Haynes 7 July 2011

Approved by: Diana Haynes Date

Deputy Director, Monitoring Programs Division

8609 Sudley Road, Suite 206, Manassas, VA 20110

(703) 330-2300 x134

ORIGINAL SIGNATURE PAGE MAINTAINED BY USDA, AMS, SCIENCE & TECHNOLOGY, MONITORING PROGRAMS DIVISION ELECTRONICALLY REPRODUCED SIGNATURES
United States Department of Agriculture

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Microbiological Data Program

SOP No: MDP-MTH-11 Page 16 of 16

Title: Real-time PCR Detection of Shiga-toxin Producing Escherichia coli (STEC), Serotype 

O157 and non-O157 in Fresh Produce and Food with non-O157 Isolation and Identification

Revision: 01 Replaces: 7/1/2010 Effective: 9/1/2011

Revision 01 June 2011 Monitoring Programs Division

 Revised SOP Title to read “...with non-O157 Isolation and Identification”

 Updated References, Section 6

 Added VITEK® and cards information, Section 7.1

 Replaced “mEC+n” with “mBPWp (with ACV added immediately)” entire SOP

 Replaced “CHROMagerTM E. coli plates” with “chromagenic agar plates”, entire SOP

 Deleted italicized “SmartMix “note at end of Section 7.2

 Increased volumes from “...4 μl of Master Mix and 1 μl of...” to “...20 μl of Master Mix 

and 5 μl of...”, Section 7.3.1

 Deleted “SmartMixTMHM”, Section 7.4.2.1

 Increased volume from “...4 μl of Master Mix...” to “...20 μl of Master Mix...”, Section 

7.4.2.2

 Deleted old Section 7.4.2.3

 Increased volume from “4 μl” to “20 μl” and increased volume from “1 μl” to “5 μl”, old 

Section 7.4.2.4

 Deleted “1:10”, changed “diluted” to “undiluted”, and added “DNA”, old Section 7.4.2.4

 Replaced the word “suspect” with “preliminary”, Section 7.6.1

 Deleted the words “2 separate” and “one” and “35±2°C and the second at”, Section 7.7.2

 Added Notes, Sections 7.7.3 and 7.7.4

 Added bold words in “Examine plates and pick at least 20 typical isolated colonies or 

swipes from any of the selective agar plates (chromogenic, L-EMB, and MacConkey) to 

appropriate media (e.g., LST, BHI, etc.)”, Section 7.7.4

 Revised “Typical Colony Characteristics of pathogenic E. coli” Table to reflect 

chromagenic agar information, Section 7.7.4

 Deleted mEC+n recipe, Section 7.8

 Deleted sentence from TP broth recipe, “For double strength TP broth, follow the 

instructions above, except add 500ml of deionized water.” , Section 7.8

 Replaced word “Suspect” with “Preliminary”, section 7.9

 Revised STEC Isolation and Identification Flowchart