Number: 331957.1Effective Date: October 2017Supersedes: New

Technology Platform: 3M™ Molecular Detection Assay 2Originating Location: St. Paul, MN

Technical Bulletin

3MTM Molecular Detection Assay 2 – Salmonella Confirmation Methods

The 3M™ Molecular Detection Assay 2 – Salmonella is used with the 3M™ Molecular Detection System for the rapid and specific detection of Salmonella in enriched food, feed and food process environmental samples. The 3M Molecular Detection Assay uses loop-mediated isothermal amplification (LAMP) to rapidly amplify Salmonella target DNA with high specificity and sensitivity, and bioluminescence to help detect the amplification. Presumptive positive results are reported in real-time while negative results are displayed after the run is completed.

Samples should be confirmed per the laboratory standard operating procedures (SOP) or by following the appropriate reference method1,2,3, beginning with transfer from the 3M™ Buffered Peptone Water ISO (BPW ISO) enrichment to secondary enrichment broth(s), followed by subsequent plating and confirmation of isolates using appropriate biochemical and serological methods (Figure 1).

Overviews of confirmation protocols according to guidelines in the USFDA Bacteriological Analytical Manual (BAM), Chapter 5: Salmonella1, USDA FSIS Microbiology Laboratory Guidebook 4.052, and the ISO-65793 method are shown in Figures 2, 3, and 4, respectively. Please refer to the 3M™ Molecular Detection Assay 2 – Salmonella Product Instructions4 and the respective standard methods for details of procedures and interpretation of results.

3M™ Molecular Detection Assay 2 – Salmonella

Add BPW ISO broth to sample Add primary enrichment broth to sample

Homogenize and incubate

Transfer to secondary enrichments

Isolate colonies on Salmonella selective gear

Purify typical colonies onnon-selective agar

Biochemical/serologicalconfirmations

Homogenize and incubate according to product instructions

Run 3M™ Molecular Detection Assay 2 – Salmonella

Follow procedure to reportresults per laboratory SOP

Reference Method

Presumptiveresult

Negative

Figure 1. General confirmation protocol for the 3M™ Molecular Detection Assay 2 – Salmonella.

An example of typical colonies on Salmonella selective agars is found in figure 5.

Positive

Confirm

ation

3M™ Molecular Detection Assay 2 – Salmonella* FDA BAM

Add BPW ISO broth to sample* Add primary enrichment media to sample

Homogenize and incubate according to product instructions Incubator per FDA BAM Ch 5.

Run 3M™ Molecular Detection Assay 2 – Salmonella

Run 3M™ Molecular Detection Assay 2 – Salmonella

Transfer 1.0 mL sample to 10 mL of TT broth**

Incubate 22-26 h at 42 ± 0.2°C (high microbial load)

35 ± 2°C (low microbial load)

Streak onto BS, XLD and HE agars **

Incubate 22-26 h at 42 ± 0.2°CIncubate additional 24 h

if needed

Biochemical confirmation per FDA BAM Ch 5.

Transfer 1.0 mL sample to 10 mL of RV broth**

Incubate 22-26 h at 42 ± 0.2°C

*Refer to 3M™ Molecular Detection Assay 2 – Salmonella product

instructions for specific protocols

** FDA BAM Ch 5 indicate the use of both RV (Rappaport-Vassiliadis) and TT (Tetrathionate) as secondary enrichment as well as the use of BS (Bismuth sulfite), XLD (Xylose lysine desoxycholate) and HE (Hektoen enteric) for identification of typical Salmonella colonies. A guide for interpretation of colonies on selective agar provided by FDA can be found in Salmonella flipbook. https://www.fda.gov/downloads/Food/FoodScienceResearch/RFE/UCM517352.pdf

Figure 2. Confirmation protocol according to US FDA Bacteriological Analytical Manual, Chapter 5: Salmonella1.

3M™ Molecular Detection Assay 2 – Salmonella USDA FSIS MLG 4.09

Add BPW ISO broth to sample* Add primary enrichment media to sample

Incubate according to product instructions Incubate as per USDA FSIS MLG 4.09

Run 3M™ Molecular Detection Assay 2 – Salmonella

Run 3M™ Molecular Detection Assay 2 – Salmonella

Transfer 0.5 mL sample to 10 mL of TT broth (Hanja)**

Incubate 22-24 h at 42 ± 0.2°C

Streak onto BGS and either DMLIA or XLT-4 agars**

Incubate 18-24 h at 35 ± 2°CIncubate additional 24 h

if needed

Biochemical confirmation per USDA FSIS MLG 4.09

Transfer 1.0 mL sample to 10 mL of RV broth**

Incubate 22-26 h at 42 ± 0.2°C

*Refer to 3M™ Molecular Detection Assay 2 – Salmonella product

instructions for specific protocols

** USDA FSIS MLG 4.09 indicate the use of both RV (Rappaport-Vassiliadis) and TT (Tetrathionate) as secondary enrichment as well as the use of BGS and an additional selective agar, DMLIA or XLT-4, for identification of typical Salmonella colonies. BGS (Brilliant green sulfa agar), DMLIA (double modified lysine iron agar), XLT-4 (Xylose lysine tergitolTM 4 agar), TSI (Triple sugar iron agar), LIA (lysine iron agar).

Figure 3. Confirmation protocol according to USDA FSIS Microbiology Laboratory Guidebook 4.092.

3M™ Molecular Detection Assay 2 – Salmonella ISO 6579-1:2017

Add BPW ISO broth to sample* Add primary enrichment media to sample

Incubate according to product instructions Incubate as per ISO 6579:2017

Run 3M™ Molecular Detection Assay 2 – Salmonella Transfer 1.0 mL sample to

10 mL of MKTTn broth**Incubate 21-27 h at 37°C

Streak onto XLD agarsIncubate 21-27 h at 37°C

Streak on a second selective plate in accordance with

manufacturer’s instructions**

Sub-culture typical colonies onto non-selective agar

Incubate 21-27 h at 37°C

Biochemical/serological confirmation per ISO 6579-2017

Transfer 0.10 mL sample to 10 mL of RV broth**

Incubate 21-27 h at 41.5°C

*Refer to 3M™ Molecular Detection Assay 2 – Salmonella product

instructions for specific protocols

** ISO 6579-1:20173 method indicates the use of both RVS (Rappaport-Vassiliadis soya peptone) and MKTTn (Muller-Kauffmann tetrathionate novobiocin) as secondary enrichment as well as the use of XLD (Xylose lysine desoxycholate) and an additional selective agar. Refer to Table 1 for a list of Salmonella selective agars.

Figure 4. Confirmation protocol according to ISO 6579-1:20173.

!

E. coli colonyon XLD agar

(yellow)

Typical Salmonella colony on XLD

agar (black)

Typical Salmonella

colonies on XLT-4 agar (black) E.coli would

be yellow

Typical Salmonella

colonies on XLT-4 agar (black) E.coli would

be yellow

Typical Salmonella colony BGA agar (pink)

E. coli would be yellow.

Figure 5. Typical colonies of Salmonella in selective agar.

Table 1. Additional Salmonella selective agars.

References:1. US Food and Drug Administration Bacteriological Analysis Manual. Chapter 5: Salmonella. August 2016 Version.2. US Department of Agriculture (USDA) FSIS Microbiology Laboratory Guidebook 4.09. Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Catfish and Siluriformes (fish) Products and Carcass and Environment Sponges. Effective Date: 01/02/2017.3. ISO 6579:2017. Microbiology of Food and Animal Feeding Stuffs – Horizontal Method for the Detection, enumeration and serotyping of Salmonella spp. Part 1: Detection of Salmonella spp.4. Product instructions: 3M™ Molecular Detection Assay 2 – Salmonella., https://multimedia.3m.com/mws/media/1129363O/mda-2-salmonella-product-instructions.pdf

Salmonella selective agar name Supplier

BLL™ Chromagar™ BD Diagnostic Systems

RAPID Salmonella Bio-Rad Laboratories S.A.

chromID Salmonella bioMérieux

Rambach™ Agar CHROMagar

CHROMagar™ Salmonella Plus CHROMagar

Salmonella Chomogenic Agar CONDA, S.A.

Harlequin™ Salmonella ABC Lab M

Chromatic™ Salmonella Liofilchem s.r.l.

Harlequin™ Chromogenic Agar for Salmonella Esterase (CASE)

Neogen Europe

HiCrome™ Salmonella Agar Sigma Aldrich

Oxoid Brilliance™ Salmonella Agar Thermom Scientific

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