Rapid and Sensitive Detection of Salmonella in Chickens Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick

Abstract

Salmonellosis is a highly contagious bacterial disease that threatens both human and poultry health. Tests that can detect Salmonella in the field are urgently required to facilitate disease control and for epidemiological investigations. Here, we combined loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) to rapidly and accurately detect Salmonella. LAMP primers were designed to target the Salmonella invA gene. LAMP conditions were optimized by adjusting the ratio of inner to outer primers, MgSO 4 concentration, dNTP mix concentration, amplification temperature, and amplification time. We evaluated the specificity of our novel LAMP-LFD method using six Salmonella species and six related non-Salmonella strains. All six of the Salmonella strains, but none of the non-Salmonella strains, were amplified. LAMP-LFD was sensitive enough to detect concentrations of Salmonella enterica subsp. enterica serovar Pullorum genomic DNA as low as 89 fg/µl, which is 1,000 times more sensitive than conventional PCR. When artificially contaminated feed samples were analyzed, LAMP-LFD was also more sensitive than PCR. Finally, LAMP-LFD gave no false positives across 350 chicken anal swabs. Therefore, our novel LAMP-LFD assay was highly sensitive, specific, convenient, and fast, making it a valuable tool for the early diagnosis and monitoring of Salmonella infection in chickens.

Rapid Detection of Escherichia coli O157:H7 by Loop-Mediated Isothermal Amplification Coupled with a Lateral Flow Assay Targeting the z3276 Genetic Marker

“…Labeled primers produce labeled amplification products, which are trapped by a biotin ligand and bound to an LF test strip. In recent studies, LAMP-LF has gained recognition in the detection of various microorganisms and substances (Liu et al 2019;Ledlod et al 2020;Allgöwer et al 2020;Jalandra et al 2020), but the use of this technology to accurately detect E. coli of serotype O157:H7 has not been reported to date. In this study, based on the z3276 genetic marker, we developed a z3276-LAMP-LF assay, combining the LAMP method with an LF test strip, which enabled faster and more specific identification of E. coli O157:H7 with a lower detection limit than previously established methods.…”

Establishment and Application of Polymerase Spiral Reaction Amplification for Salmonella Detection in Food

“…Currently, the classical official methods for Salmonella detection and identification include mainly bacterial isolation and biochemical identification, which are timeconsuming and can seriously delay the acquisition of detection results [30]. Although the PCR-based methods have been successfully established as a valuable method for Salmonella detection, their complicated procedures and expensive equipment, especially for the PSR method, decrease their suitability for extensive practical use [6,31].…”

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

“…Many previous reports published only a single LFB assay for the detection of either Campylobacter or Salmonella spp. in food [37][38][39], as it is challenging to enrich both microorganisms simultaneously, due to the fact that their growth requirements on different selective broths as well as slower growth of Campylobacter under microaerobic conditions [40]. However, as previously described [41], both organisms contaminated in food were enriched independently and were then gathered for DNA extraction.…”

“…However, the analytical sensitivity of our d-LAMP-LFB assay in spiking study was also comparable to the LFB in other previous studies. It has been reported that the lowest inoculated detection limit for Salmonella targeting invA gene was 8×10 3 CFU/25 g (320 CFU/g) after enrichment for 18-24 h [37], 36 CFU/25 g (1.44 CFU/g) targeting hilA gene after 6 of enrichment [38], while C. jejuni was 10 1 −10 2 CFU/ 25 g (or 10 1 −10 2 CFU/mL of initial inoculum levels) targeting hipO gene without enrichment step [39]. Several qPCR kits such as BAX 1 Campylobacter coli/jejuni/lari assay (DuPont Qualicon), BAX 1 Salmonella assay (Hygiena), BIOTECON foodproof 1 Campylobacter Detection Kit (Biotecon Diagnostics) and BIOTECON foodproof 1 Salmonella Detection Kit (Biotecon Diagnostics) are commercially available with the level of detection as low as 1-10 CFU/25 g sample or 10 3 −10 4 CFU/mL after 20-24 h and 24-48 h of enrichment for Salmonella and Campylobacter, respectively.…”