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KMID : 0545120190290030454
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Journal of Microbiology and Biotechnology
2019 Volume.29 No. 3 p.454 ~ p.464
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Rapid and Sensitive Detection of Salmonella in Chickens Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick
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Liu Zhi-Ke
Zhang Qiu-Yu
Yang Ning-Ning
Xu Ming-Guo
Xu Jin-Feng
Jing Ming-Long
Wu Wen-Xing
Lu Ya-Dong
Shi Feng
Chen Chuang-Fu
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Abstract
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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, MgSO4 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.
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KEYWORD
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Salmonella, invA gene, loop-mediated isothermal amplification, lateral flow dipstick, detection
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