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.

Keywords: LAMP; Salmonella; animal food; multi-laboratory; validation.

FIGURE 1

A schematic diagram of the MLV study design comparing the LAMP alternative method and the FDA BAM Chapter 5 reference method for the detection of Salmonella Infantis ATCC 51741 in 25 g dry dog food test portions. RV, Rappaport-Vassiliadis medium; TT, tetrathionate broth; BS, bismuth sulfite agar; XLD, xylose lysine desoxycholate agar; HE, Hektoen enteric agar; TSI, triple sugar iron agar; LIA, lysine iron agar.

FIGURE 2

Sample analysis flowchart for the MLV study. LB, lactose broth; BPW, buffered peptone water; RV, Rappaport-Vassiliadis medium; TT, tetrathionate broth; BS, bismuth sulfite agar; XLD, xylose lysine desoxycholate agar; HE, Hektoen enteric agar; TSI, triple sugar iron agar; LIA, lysine iron agar; qPCR, real-time quantitative PCR; MALDI, Matrix Assisted Laser Desorption Ionization.

FIGURE 3

Representative LAMP graphs and results generated and viewed using the Genie Explorer software (version 2.0.6.3). (A) Amplification; (B) amplification rate; (C) anneal; (D) anneal derivative; and (E) results. Samples with three-digit codes correspond to dry dog food test portions with low-level inoculation of Salmonella Infantis ATCC 51741. Samples marked as PC are positive control, i.e., S. enterica Typhimurium ATCC 19585 (LT2) at 1.7 × 10⁴ CFU/reaction. Samples marked as NTC are no template control, i.e., molecular grade water.