Modified Most Probable Number Assay to Quantify Salmonella in Raw and Ready-to-Cook Chicken Products

Summary

Accurately quantifying Salmonella in poultry at low levels is a current industrial and regulatory challenge. This protocol describes an MPN assay that enables quantification of Salmonella in raw and ready-to-cook poultry products. This method is fast, sensitive, and aligns with FSIS guidelines, enhancing food safety and supporting public health efforts.

Abstract

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella focus on prevalence rather than quantification, which limits their utility in assessing contamination levels and risks. This study introduces a novel most probable number (MPN) assay designed to quantify Salmonella in ready-to-cook poultry products, such as chicken cordon bleu. The method involves washing the poultry sample, concentrating the rinse through centrifugation, and serially diluting it in a 48-well block. The MPN assay is integrated with the loop-mediated isothermal amplification (LAMP) method to provide a sensitive, accurate, and rapid quantification of Salmonella contamination within the same timeframe as existing Food Safety and Inspection Service (FSIS) protocols. Results show a strong linear correlation between the MPN-LAMP measurements and theoretical inoculation levels (R² = 0.933). However, variability at lower concentrations highlights challenges in accurately detecting Salmonella at these levels, with the practical lower limit of detection estimated at approximately 300 CFU/g. Potential refinements to improve the protocol's applicability include increasing the quantity sampled to further improve the limit of detection, optimizing enrichment media formulations, and expanding molecular detection to target multiple Salmonella serovars. Overall, this study presents a practical tool for the food industry, enabling reliable quantification of Salmonella contamination in poultry products, contributing to improved food safety and public health.

Introduction

As a leading cause of foodborne illness, hospitalization, and death in the U.S., Salmonella has a significant public health and economic impact. The pathogen's estimated economic burden in 2013 alone was $3.67 billion¹. Although recent regulatory initiatives aim to reduce salmonellosis by 25% by 2030², gaps in current detection and mitigation strategies remain evident, particularly in aligning processing plant surveillance with public health outcomes³ .

Frozen ready-to-cook poultry products, which have been implicated in multiple Salmonella outbrea....

Protocol

NOTE: All work associated with this protocol should be conducted within a Biosafety Level 2 (BSL-2) laboratory. When appropriate, this protocol should be conducted within a biological safety cabinet (BSC) to maintain aseptic conditions and minimize the risk of sample contamination or operator exposure to microbial pathogens. When transferring samples outside the BSC, use sealed containers to maintain sample integrity and prevent spillage in case of accidental drops. Preferably, disposable components should be used throughout the procedure to mitigate the possibility of cross-contamination. In cases where disposables are not feasible, ensure all equipment and materials....

Discussion

Significance of the protocol
Salmonella remains a major concern in food safety, particularly within poultry products, which are often implicated in foodborne illness outbreaks^13,14. As a leading cause of bacterial foodborne illness in the United States, reliable methods for detecting Salmonella in both fresh and ready-to-cook poultry products are critical to ensuring food safety¹⁵. The ability to qu.......

Materials

Name	Company	Catalog Number	Comments
48 deep well block 4.6ml	Fisher Scientific International, Inc	NC1964628
Agar - Solidifying Agent (Difco)	Becton, Dickinson and Company (BD)	281230
Analytical Balance	Mettler Toledo	JL602-G/L	Equipment
Analytical Balance	Mettler Toledo	AB54-S	Equipment
Autoclave - Amsco Lab250, Laboratory Steam Sterilizer	Steris plc	LV-250	Equipment
Biological Safety Cabinet, Type A2, Purifier Logic+	Labconco Corporation	302411101	Equipment
Brain Heart Infusion (BHI) Broth	Becton, Dickinson and Company (BD)	237500
Buffered Peptone Water	Bio-Rad Laboratories Inc.	3564684
Cell Spreader - L-shaped	VWR	76208-438
Centrifuge Microcentrifuge 5424	Eppendorf	5424	Equipment
Centrifuge, Avanti J-25	Beckman Coulter, Inc.		Equipment
DNA Extraction - PreMan Ultra Sample Preparation Reagent	Thermo Fisher Scientific Inc.	4318930
Ground Chicken	Local retailers
IAC forward  primer: 5'-GGCGCGCCTAACACATCT-3'	Integrated DNA Technologies
IAC probe: 5'-TTACAACGGGAGAAGACAATGC CACCA-3' labeled with 5' TAMRA/3' BHQ-2	Biosearch Technologies
IAC reverse primer: 5'-TGGAAGCAATGCCAAATGTGTA-3'	Integrated DNA Technologies
Incubator - Inova 4230 incubator shaker	New Brunswick Scientific	4230	Equipment
Inoculating Loop - Combi Loop  10µL and 1µL	Fisher Scientific International, Inc	22-363-602
invA forward primer: 5'-GTTGAGGATGTTATTCGCAAAG G-3'	Integrated DNA Technologies
invA probe: 5'-CCGTCAGACCTCTGGCAGTAC CTTCCTC-3' labeled with 5' Cal Fluor Orange 560/3' BHQ-1	Biosearch Technologies
invA reverse primer: 5'-GGAGGCTTCCGGGTCAAG-3'	Integrated DNA Technologies
Irradiation Treatment	Texas A&M Agrilife Research National Center for Electron Beam Research		Service
Luria Bertani (LB) Broth	Becton, Dickinson and Company (BD)	244620
Manual pipette Pipet-Lite LTS Pipette L-1000XLS+	Mettler Toledo	17014382	Equipment
Manual pipette Pipet-Lite LTS Pipette L-100XLS+	Mettler Toledo	17014384	Equipment
Manual pipette Pipet-Lite LTS Pipette L-10XLS+	Mettler Toledo	17014388	Equipment
Manual pipette Pipet-Lite LTS Pipette L-200XLS+	Mettler Toledo	17014391	Equipment
Manual pipette Pipet-Lite LTS Pipette L-20XLS+	Mettler Toledo	17014392	Equipment
Manual pipette Pipet-Lite Multi Pipette L8-200XLS+	Mettler Toledo	17013805	Equipment
Manual pipette Pipet-Lite Multi Pipette L8-20XLS+	Mettler Toledo	17013803	Equipment
Media Storage Bottle -PYREX 1L Square Glass  Bottle, with GL45 Screw Cap	Corning Inc.	1396-1L	Equipment
Media Storage Bottle -PYREX 2L Round Wide Mouth Bottle, with GLS80 Screw Cap	Corning Inc.	1397-2L	Equipment
Microtiter plate, 96 well plate, flat bottom, polystyrene, 0.34cm2, sterile, 108/cs	MilliporeSigma	Z707902
Mixer - Vortex Genie 2	Scientific Industries Inc.	SI-0236	Equipment
Molecular Detection Assay 2-Salmonella kit	Neogen	MDA2SAL96
Molecular Detection Instrument	Neogen	MDS100	Equipment
Motorized pipette controller, PIPETBOY2	INTEGRA Biosciences Corp.	155019	Equipment
PCR Mastermix 2× TaqMan Gene Expression	Thermo Fisher Scientific Inc.	4369542
Petri Dish Rotator -  bioWORLD Inoculation Turntable	Fisher Scientific International, Inc	3489E20	Equipment
Petri Dishes with Clear Lid (100 mm x 15mm)	Fisher Scientific International, Inc	FB0875713
Pipette Tips GP LTS 1000µL S 768A/8	Mettler Toledo	30389273
Pipette Tips GP LTS 20µL 960A/10	Mettler Toledo	30389270
Pipette Tips GP LTS 200µL F 960A/10	Mettler Toledo	30389276
Ready to cook chicken products	Local retailers
Reagent Reservoir, 25 mL sterile reservoir used with multichannel pipettors	Thermo Fisher Scientific Inc.	8093-11
Realtime PCR - 7500 Real-Time PCR system	(Applied Biosystems, Foster City, CA)	2750036476	Equipment
Serological Pipettes, Nunc Serological Pipettes (10 mL)	Thermo Fisher Scientific Inc.	170356N
Serological Pipettes, Nunc Serological Pipettes (2 mL)	Thermo Fisher Scientific Inc.	170372N
Serological Pipettes, Nunc Serological Pipettes (25 mL)	Thermo Fisher Scientific Inc.	170357N
Serological Pipettes, Nunc Serological Pipettes (50 mL)	Thermo Fisher Scientific Inc.	170376N
Spreader - Fisherbrand L-Shaped Cell Spreaders	Fisher Scientific International, Inc	14-665-230
Stomacher bag, Nasco Whirl-Pak Write-On Homogenizer Blender Filter Bags	Thermo Fisher Scientific Inc.	01-812
Stomacher 80 Biomaster Lab Blender	Seward	30010019	Equipment
Thermocycler (GeneAmp PCR system 9700)	Applied Biosystems	535028293	Equipment
Water Filtration - Elga Veolia Purelab Flex	Elga LabWater	PF2XXXXM1-US	Equipment
Whirlpak bags 1.63L	VWR	11216-777