Rapid and simultaneous detection of Campylobacter spp. and Salmonella spp. in chicken samples by duplex loop-mediated isothermal amplification coupled with a lateral flow biosensor assay.

Thanawat Sridapan,Wansika Kiatpathomchai,Taradon Luangtongkum,Surang Chankhamhaengdecha,Wanida Tangkawsakul,Tavan Janvilisri,Arun K Bhunia

doi:10.1371/journal.pone.0254029

Abstract

Development of a simple, rapid and specific assay for the simultaneous detection of Campylobacter spp. and Salmonella spp. based on duplex loop-mediated isothermal amplification (d-LAMP), combined with lateral-flow biosensor (LFB) is reported herein. LAMP amplicons of both pathogens were simultaneously amplified and specifically differentiated by LFB. The specificity of the d-LAMP-LFB was evaluated using a set of 68 target and 12 non-target strains, showing 100% inclusivity and exclusivity. The assay can simultaneously detect Campylobacter and Salmonella strains as low as 1 ng and 100 pg genomic DNA per reaction, respectively. The lowest inoculated detection limits for Campylobacter and Salmonella species in artificially contaminated chicken meat samples were 103 CFU and 1 CFU per 25 grams, respectively, after enrichment for 24 h. Furthermore, compared to culture-based methods using field chicken meat samples, the sensitivity, specificity and accuracy of d-LAMP- LFB were 95.6% (95% CI, 78.0%-99.8%), 71.4% (95% CI, 29.0%-96.3%) and 90.0% (95% CI, 73.4%-97.8%), respectively. The developed d-LAMP-LFB assay herein shows great potentials for the simultaneous detection of the Campylobacter and Salmonella spp. and poses a promising alternative approach for detection of both pathogens with applications in food products.

Highlights

Foodborne pathogens can cause serious adverse effects via contaminated food or water
Results from duplex loop-mediated isothermal amplification (d-Loop-mediated isothermal amplification (LAMP))-lateral-flow biosensor (LFB) were compared to the detection performance to those of standard culture-based method
The sensitivity, specificity and accuracy were calculated with 95% confidence intervals using MedCalc

Summary

Introduction

Foodborne pathogens can cause serious adverse effects via contaminated food or water. The number of cases of Campylobacter and non-typhoidal Salmonella was estimated at >95 and >78 million foodborne illnesses worldwide, respectively [1] Both pathogens are highly prevalent in poultry, especially commercial chicken meat, which is often implicated as the main food vehicle of infection for human through the consumption of raw or undercooked contaminated poultry meat and products [2,3,4,5]. Loop-mediated isothermal amplification (LAMP) has been widely used to overcome the drawback of those assays because it is performed under constant temperature with high sensitivity, specificity and rapidity for the low-cost detection of pathogens [11, 12] To this date, an advancement of LAMP method, namely multiplex LAMP (m-LAMP) has been increasingly applied for simultaneous detection of multiple target genes in a one-tube reaction.

Materials and methods

Results

Discussion