Upload
others
View
6
Download
0
Embed Size (px)
Citation preview
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
3M and 3M Science. Applied to Life. are trademarks of 3M. Used under license in Canada. © 2019, 3M. All rights reserved. All other trademarks are property of their respective owners. 1906-15150 E
3M Food Safety Division 3M CanadaP.O. Box 5757London, ON N6A 4T11-800-364-35773M.ca/FoodSafety