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 <i>Salmonella</i> 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 <i>Salmonella</i>. LAMP primers were designed to target the <i>Salmonella invA</i> gene. LAMP conditions were optimized by adjusting the ratio of inner to outer primers, MgSO₄ concentration, dNTP mix concentration, amplification temperature, and amplification time. We evaluated the specificity of our novel LAMP-LFD method using six <i>Salmonella</i> species and six related non-<i>Salmonella</i> strains. All six of the <i>Salmonella</i> strains, but none of the non-<i>Salmonella</i> strains, were amplified. LAMP-LFD was sensitive enough to detect concentrations of <i>Salmonella enterica</i> subsp. <i>enterica</i> 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 <i>Salmonella</i> infection in chickens.