Multi-Laboratory Validation of a Loop-Mediated Isothermal Amplification Method for Screening Salmonella in Animal Food

Abstract

Loop-mediated isothermal amplification (LAMP) has gained wide popularity in the detection of Salmonella in foods owing to its simplicity, rapidity, and robustness. This multi-laboratory validation (MLV) study aimed to validate a Salmonella LAMP-based method against the United States Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella reference method in a representative animal food matrix (dry dog food). Fourteen independent collaborators from seven laboratories in the United States and Canada participated in the study. Each collaborator received two sets of 24 blind-coded dry dog food samples (eight uninoculated; eight inoculated at a low level, 0.65 MPN/25 g; and eight inoculated at a high level, 3.01 MPN/25 g) and initiated the testing on the same day. The MLV study used an unpaired design where different test portions were analyzed by the LAMP and BAM methods using different preenrichment protocols (buffered peptone water for LAMP and lactose broth for BAM). All LAMP samples were confirmed by culture using the BAM method. BAM samples were also tested by LAMP following lactose broth preenrichment (paired samples). Statistical analysis was carried out by the probability of detection (POD) per AOAC guidelines and by a random intercept logistic regression model. Overall, no significant differences in POD between the Salmonella LAMP and BAM methods were observed with either unpaired or paired samples, indicating the methods were comparable. LAMP testing following preenrichment in buffered peptone water or lactose broth also resulted in insignificant POD differences ( P > 0.05). The MLV study strongly supports the utility and applicability of this rapid and reliable LAMP method in routine regulatory screening of Salmonella in animal food.

Rapid Screening for Salmonella in Raw Pet Food by Loop-Mediated Isothermal Amplification

“…LAMP screening. After overnight incubation at 358C in BPW or LB, aliquots (1 mL) of each test portion were removed from the filtered side of the bag, and DNA was extracted using the PrepMan ultra sample preparation reagent (Thermo Fisher Scientific, Waltham, MA (11,17)). In the LAMP assay, 2 μL of the DNA extracts was used, and the assay was repeated once independently.…”

“…The LAMP assay was conducted as described previously (10,11,17); the only exception was that a new isothermal master mix (ISO-004, OptiGene Ltd., West Sussex, UK) was used side by side with the original one (ISO-001, OptiGene). The LAMP reagent mixture in a total volume of 25 μL contained 13 isothermal master mix (ISO-001 or ISO-004), 13 primer mix (0.1 μm each of outer primer [Sal4-F3 and Sal4-B3], 1.8 μm each of inner primer [Sal4-FIP and Sal4-BIP], and 1 μm each of loop primer [Sal4-LF and Sal4-LB]; Integrated DNA Technologies, Coralville, IA), and 2 μL of DNA template.…”

“…We recently developed a rapid Salmonella screening method (10,17,51) based on an isothermal nucleic acid amplification test: loop-mediated isothermal amplification (LAMP) (30). Through the formation of a target-specific stem-loop DNA structure, LAMP uses four to six specific primers and a strand-displacing DNA polymerase to efficiently amplify the target DNA under a constant temperature (~658C).…”

“…Our Salmonella LAMP assay specifically targets the Salmonella invasion gene invA (GenBank accession M90846 (16)), which possesses a broad specificity for Salmonella serovars (33). Following successful singlelaboratory validation in six animal food matrices (10) and multilaboratory validation in dry dog food (17), the method was incorporated into the FDA's Bacteriological Analytical Manual (BAM), Chapter 5, as a Salmonella screening method in animal food and confirmation method for presumptive Salmonella isolates from all food categories (1). However, the assay has not been applied to raw pet food, which consists of a group of starkly different matrices from dry pet food.…”

Formic Acid as an Antimicrobial for Poultry Production: A Review

“…Previously, Salmonella spp. contamination of animal and poultry feed has received most of the attention and remains a current focus for the development of detection methods suited explicitly for feeds as well as the pursuit of more effective control measures (12,26,30,(45)(46)(47)(48)(49)(50)(51)(52)(53). Numerous Salmonella spp.…”