May 11, 2026

Screening Canine Feces with the MICROSEQ® Salmonella spp. Detection Kit.

DOI
https://dx.doi.org/10.17504/protocols.io.5jyl82bddl2w/v1
  • 1FDA CVM;
  • 2UPenn (retired);
  • 3Iowa State University (Retired);
  • 4Ohio Department of Agriculture (Retired);
  • 5University of Missouri;
  • 6Texas A&M Veterinary Medical Diagnostic Laboratory;
  • 7Texas A&M University - College Station;
  • 8Ohio Department of Agriculture;
  • 9University of Arizona;
  • 10FDA HFP Moffett PT;
  • 11FDA CVM (Retired)
  • Vet LIRN
Megan Miller
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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl82bddl2w/v1
External link: https://doi.org/10.1128/jcm.02137-16
Protocol Citation: Sarah Nemser, Shelley Rankin, Timothy Frana, Beverly Byrum, Shuping Zhang, Amy Swinford, Sara Lawhon, Jing Cui, Yan Zhang, Shannon Kiener, Megan Miller, Renate Reimschuessel 2026. Screening Canine Feces with the MICROSEQ® Salmonella spp. Detection Kit.. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82bddl2w/v1
Manuscript citation:
Reimschuessel R, Grabenstein M, Guag J, Nemser SM, Song K, Qiu J, Clothier KA, Byrne BA, Marks SL, Cadmus K, Pabilonia K, Sanchez S, Rajeev S, Ensley S, Frana TS, Jergens AE, Chappell KH, Thakur S, Byrum B, Cui J, Zhang Y, Erdman MM, Rankin SC, Daly R, Das S, Ruesch L, Lawhon SD, Zhang S, Baszler T, Diaz-Campos D, Hartmann F, Okwumabua O. Multilaboratory Survey To Evaluate Salmonella Prevalence in Diarrheic and Nondiarrheic Dogs and Cats in the United States between 2012 and 2014. J Clin Microbiol. 2017 May;55(5):1350-1368. doi: 10.1128/JCM.02137-16. Epub 2017 Feb 15. PMID: 28202802; PMCID: PMC5405253.
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 02, 2024
Last Modified: May 11, 2026
Protocol  Integer ID: 102728
Keywords: Salmonella, veterinary diagnostics, animal feces, screening canine fece, grant evaluation of salmonella, salmonella spp, study for the vet, salmonella, asymptomatic pet, participating vet, detection kit, vet, pcr method, detection
Funders Acknowledgements:
Vet-LIRN
Grant ID: RFA-FD-11-010
Disclaimer
Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Abstract
The PCR method was developed and validated by 11 participating Vet-LIRN laboratories as a collaborative effort under the grant Evaluation of Salmonella in Symptomatic and Asymptomatic Pets: Study for the Vet-LIRN Program (https://grants.nih.gov/grants/guide/rfa-files/rfa-fd-11-010.html).




Guidelines
Sample submission
Feces should be received fresh or on ice in individually marked containers. At least 1 gm of feces is required and 10-25 gm of feces is preferred. Frozen samples or fecal swabs are not acceptable. Samples should be set-up in pre-enrichment within 4 hours of receipt. If a sample is to be used for additional testing, then the sample should be split as needed and the results reported separately.
Materials
Critical consumables and sources include:
ABC
Item DescriptionVendorCatalog Number
Buffered Peptone Water, 500g eachRemelR452672
MagMAX™ Total Nucleic Acid Isolation Kit, 100 preps Thermo Fisher Scientific AM1840
PrepMan™ Ultra Sample Preparation Reagent, 200 prepsThermo Fisher Scientific 4318930
MicroSEQ™ Salmonella spp. Detection Kit, 96 assaysThermo Fisher Scientific 4403930
KingFisher 96 microplate, 48/case Thermo Fisher Scientific 97002540
KingFisher 96 deep-well plate, 50/caseThermo Fisher Scientific 95040450
KingFisher 96 tip comb for deep-well magnets, 100/caseThermo Fisher Scientific 97002534

Standard Equipment and Materials for MicroSeq‱ PCR Protocol
  • Whirl-Pak‱ bags or equivalent
  • Disposable gloves
  • Incubators, 37 ± 1 ºC
  • Balance
  • Racks for Whirl-Pak‱ bags
  • Tongue depressors
  • Pipettes and pipette tips (1000, 200, 100, 10μl)
  • Microcentrifuge
  • Vortex
  • Vortex adaptor
  • KingFisher Duo or Flex (or other extraction method)
  • 100% Isopropanol
  • 100% Ethanol
  • 96 well FAST Optical PCR plates or Optical FAST PCR 8 tube strip
  • Optical Film or Optical PCR 8 cap strips
  • Applied Biosystems 7500 Fast Real Time PCR System
  • Laminar Flow PCR Cabinet
  • Nuclease free H20
  • RapidFinder Express Software
  • 7500 v2.3 Software
  • -20 °C freezer
  • External flash drive



Pre-enrichment in non-selective liquid medium
Using a tongue depressor, aseptically weight 10 g of fecal sample into a Whirl-Pak‱ bag

Add 90 mL of ambient temperature buffered peptone water (BPW)

Mix the fecal sample in the bag.  Use the necessary quantity of pre-enrichment medium to yield a 1/10 dilution with feces
Incubated at 37 °C ± 1 °C for 18 h ± 2 h.
Automated DNA Extraction - MagMAX Total Nucleic Acid Isolation Kit (KingFisher Duo/Flex Required)
MagMAX Total Nucleic Acid Isolation Kit manufacture protocol Download MagMAX™ Total Nucleic Acid Isolation Kit Manufacturer Protocol.pdfMagMAX™ Total Nucleic Acid Isolation Kit Manufacturer Protocol.pdf385.1KB

Preparation of solutions
Add 12 mL 100% Isopropanol to the bottle labeled “Wash Solution 1 Concentrate”

Add 32 mL 100% Ethanol to the bottle labeled “Wash Solution 2 Concentrate”

Add 360 µL Carrier NA to the bottle labeled “Lysis/Binding Solution Concentrate”; Label as “Lysis/Binding Solution” (this is stable at room temperature for 1 month)

Add 1100 µL NA Binding Beads to a 15 ml tube.  Add 1100 l Lysis/Binding Enhancer to the 15 ml tube; Dispense 1000 μL into 1.5ml tubes.  Label as “Bead Mix” (this is stable at 4 º C for 2 weeks)

Dispense 200 µL Lysis/Binding Solution into a Bead Tube for each unknown sample

Add300 µL of the pre-enriched fecal sample to each Bead Tube

Leave Whirl-Pak‱ bags at room temperature until PCR results are available
Vortex unknown samples using Vortex adapter for 15 minutes at highest setting (caps should face the center of the adapter)
Centrifuge at 16,000 rcf for 3 min
Preparation of Plates for KingFisher Duo or Flex
Sample Plate
  • Label 1 Kingfisher 96 DW plate as “Sample"
  • Add 20 µL of Bead Mix to the wells containing the unknown samples
  • Carefully add 300 µL of pre-enriched fecal sample supernatant to each well containing bead mix. Do not disturb pellet in Bead Tube.
  • Add 150 µL of 100% Isopropanol to the wells containing the unknown samples



Wash 1 Plate
  • Label 2 Kingfisher 96 DW plates as “Wash 1”
  • Add 300 µL of Wash Solution 1 to the wells corresponding to those containing unknown samples from the sample plate

Wash 2 Plate
  • Label 2 Kingfisher 96 DW plates as “Wash 2”
  • Add 450 µL of Wash Solution 2 to the wells corresponding to those containing unknown samples from the sample plate

Elution Plate
  • Label 1 96 well plate as “Elution”
  • Add 75 µL of Elution Buffer to the wells corresponding to those containing unknown samples from the sample plate

Tip Plate
  • Place one 96 well DW tip comb onto a Kingfisher 96 200 ul plate
  • Take caution not to contaminate the surface of the tip comb
Dilution Plate
  • Label 1 96 well plate as “Dilution”
  • Add 90 µL of Nuclease Free H20 to the wells corresponding to those containing unknown samples from the sample plate

Set up KingFisher Program according to the KingFisher Duo or Flex Protocol MagMax Pathogen Standard Volume
KingFisher Protocols can be found on ThermoFisher website: https://www.thermofisher.com/order/catalog/product/AM1840
Protocol requires Pathogen High Volume program
Load KingFisher instrument with the Wash, Sample, and Elution plates as directed by the machine protocol
KingFisher equipment will give an estimated completion time for protocol once loaded
Preparing the DNA
Transfer 10 ul of unknown sample from the Elution plate to the Dilution plate

The DNA is 1:10 dilution and ready for PCR
Manual DNA Extraction using PrepSeq Kit
Download PrepMan Ultra Manufacturers Protocol.pdfPrepMan Ultra Manufacturers Protocol.pdf464.3KB

Remove 300 µL fecal sample to a 1.5 ml tube; centrifuge at highest speed for 3 min and discard supernatant.

Resuspend the pellet in100 µL PrepMan Ultra Sample Preparation Reagent

Incubate at 100°C for 10 min; centrifuge at highest speed for 3 minutes

Dilution of DNA - automated extraction recommends starting with a 1:10 dilution. If inhibitors are suspected, subsequent serial dilutions may be made.

For a 1:10 dilution, Transfer5 µL of the supernatant to a 1.5 mL tube and add 45 µL nuclease free water.
For a 1:100 dilution, Transfer5 µL of the supernatant to a 1.5 mL tube and add 495 µL nuclease free water. This is the historical option for manual DNA extraction, if this is what has worked in the past, you may go directly to a 1:100 dilution.
Use immediately or store at -20°C until use.
PCR Assay- MicroSeq® Salmonella spp.
PCR Assay- MicroSeq® Salmonella spp. manufacture protocol Download MicroSEQ Salmonella PCR Detection Kit Manufacturers Protocol.pdfMicroSEQ Salmonella PCR Detection Kit Manufacturers Protocol.pdf706.4KB

Preparing the assay beads
Remove the appropriate number of individual tubes or 8-tube strips, one tube for each reaction that you plan to run.
Gently remove, then discard the colored caps. Avoid disturbing the beads from the bottom of the tubes.
Add25 µL of Nuclease free H20 to each tube

For each unknown sample or control, transfer 5 µL of the 1:10 diluted DNA to each tube

Cap the tubes, sealing each tube with the transparent optical strip caps provided in the kit, using the MicroAmp‱ 96-Well Base and the MicroAmp‱ Cap Installing Tool if necessary.
Confirm that the strips are straight and that each tube is in line with the adjacent tube.
Run samples using RapidFinder Express OR 7500 v2.3 software.
PCR Assay - RapidFinder Express OR 7500 v2.3
Open RapidFinder Express Software
Select a Run File Option specific to project
Enter Run File Information
Select Target Check
  • Enter the number of unknown samples
  • Enter at least 1 for the # of negative controls for each assay
  • Enter at least 1 for the # of positive controls for each assay
Enter Unknown sample Names

Confirm Run Layout
Start Instrument Run
7500 Software v2.3
Program the ABI 7500-FAST machine with the following settings (use fast mode):

ABCD
Run ParametersRepsTemperatureDuration (sec)
Holding Stage195 C20
Cycling Stage 14095 C03
Cygling Stage 24060 C30

Place Samples in ABI-7500 FAST thermocycler using the tube strip adapter. Balance the tubes on
each side according to the manufacturer’s instructions (blank tubes may be used for this purpose
if necessary). If you do not have this adapter or are using a different machine, you can transfer
the reactions into a plate that is compatible with your setup if needed.
Start the run.
Following run, analyze the results using the automatic analysis setting. All samples should have a
VIC (internal control) signal. If the VIC is >35 or undetermined, repeat the PCR using a 1:5 dilution
of the sample in nuclease-free water and interpret based on the result that gives the lowest Ct for
the VIC.
Set the Threshold by selecting the Positive Control wells, switch Graph Type to Linear, and manually place Threshold bar at the start of the exponential curve.
Save a copy of the original run file and export the data into an Excel file. Interpret the results
according to the following guidelines:

ABCD
FAMaVICbInterpretationAction
undetermineddetectedNot DetectedNone - this result is final
< 35disregardPositiveCulture Confirmation is optional
≥ 35detectedSuspectCulture Confirmation is optional
undeterminedundeterminedInconclusiveCulture Confirmation is optional

Once complete keep extracted undiluted and diluted DNA samples at -20C for possible follow up
Protocol references
ISO 6579:2002 Annex D- “Detection of Salmonella spp in animal feces and in samples of the primary production stage

KingFisher Protocols can be found on ThermoFisher website: https://www.thermofisher.com/order/catalog/product/AM1840"

RapidFinder Software:
RapidFinder‱ Express Software | Thermo Fisher Scientific - US