Establishment and application of a loop-mediated isothermal amplification assay for the detection of Salmonella in feces of tree shrews / 中国比较医学杂志

ABSTRACT

Objective To Establish a loop-mediated isothermal amplification(LAMP)assay for detection of Salmonella in fecal samples of tree shrews, and report the result of preliminary application of this method. Methods LAMP primers were designed and synthesized according to the conserved sequence of Salmonella specific gene invA (invasive protein gene A). To optimize the reaction time and temperature by setting 10 reaction times(24 to 42 min)and temperature(57℃ to 66℃)and tested its specificity and sensitivity. At the same time, a conventional PCR test was performed to verify and compare with the LAMP assay. 91 fecal samples of wild-derived tree shrews were detected by the LAMP assay. Results The experimental condition was confirmed as 62℃ and 34 min. The sensitivity of Salmonella was 3.36×101CFU/mL, which was 10 to 100 times higher than that of conventional PCR assay. In 10 kinds of intestinal bacteria for LAMP amplification,Salmonella enteritidis and Salmonella paratyphi B were positive,the others were negative. Among the 91 samples of tree shrew fecal samples detected by the LAMP assay, the positive detection rate was 20.88%. The LAMP assay can be completed within 40 min, the result can be observed and judged visually by color changes. Conclusions The LAMP assay established in this study is convenient,rapid,sensitive and specific. It can be used as a rapid measure for large-scale detection of Salmonella in feces of tree shrews.