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

Beilei Ge,Guodong Zhang,Dancia Wu,Ju Sheng,Xiaokuang Lai,Qianru Yang,Azadeh A Yousefvand,Qiu You,Sammie P La,Kelly J Domesle,Kirsten A Hirneisen,Thomas S Hammack,Tameji Eames,Shizhen S Wang,Yuan Hu,Lijun Hu,Jan Ryan Dollete,Cynthia Logie,Richelle S Richter,Ruiqing Pamboukian,Kyson X Chou,Cindy H Wu,Xiaohong Deng,Paul K Park,Lisa Ledger,Shenia Young ,David T Kiang ,Hugh Y Cai ,L Hahn ,Ðurđa Slavić ,Sherry Ayers ,R Diyo

doi:10.3389/fmicb.2019.00562

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.

Highlights

Salmonella is a ubiquitous human and animal pathogen, with human outbreak-related illnesses broadly attributed to multiple food categories of plant and animal origins (Interagency Food Safety Analytics Collaboration [IFSAC], 2018)
We previously developed a loop-mediated isothermal amplification (LAMP) assay targeting the Salmonella invasion gene invA and showed it to be rapid, reliable, and robust in multiple food matrices (Chen et al, 2011; Yang et al, 2013, 2014, 2015, 2016; Domesle et al, 2018)
Salmonella most probable number (MPN) obtained in the two inoculated sample sets, with a 95% confidence interval, were 0.65 MPN/25 g (0.30, 1.40) for the low level and 3.01 MPN/25 g (1.31, 6.89) for the high level

Summary

Introduction

Salmonella is a ubiquitous human and animal pathogen, with human outbreak-related illnesses broadly attributed to multiple food categories of plant and animal origins (Interagency Food Safety Analytics Collaboration [IFSAC], 2018). Two distinct advantages of LAMP over PCR are running at a constant temperature (Notomi et al, 2000) and high tolerance to matrix inhibitors (Kaneko et al, 2007), which obviate the need for a sophisticated thermocycler or a complicated DNA extraction protocol. These attractive features have led to the development of many new Salmonella LAMP assays, portable microfluidic devices, and commercially available systems (Yang et al, 2018)

Methods

Results

Conclusion