Study On LAMP Method Of Stn Gene For Rapid Detection Of Salmonella

Salmonella is a kind of pathogen to human and animal can cause disease. according to World Health organization(WHO) reported since twentieth Century, a growing threat of Salmonella to human health has attracted the attention of WHO. In recent years, with import and export trade growing prosperity, It also makes the global spread of Salmonella possible. so there are a lot of work need to be done by the worker of port inspection, Therefore, establish a fast and accurate pathogenic microorganism detection system is particularly important.Objective:This study intends to make the salmonella enterotoxin gene (stn) as a target and establish loop-mediated isothermal amplification of DNA for salmonella detection, to establish a feasible rapid salmonella detection technology, improve the detection rate of salmonella, and shorten the testing time, to provide timely and accurate diagnosis basis for salmonella detection and to improve the detection rate of salmonella. We hope to provide technical support for port inspection, monitoring of infectious diseases and prevention and control of salmonella infection. Methods:1We retrieved the stn gene nucleotide sequences in GenBank database and designed corresponding LAMP primers by PrimerExplorerV4. Oligo6.0was implied to ensure that no dimer formation between primers. At last we got4specific primers through comparing the homology of primers and amplification products in GenBank by Blast.2We used Bst with chain replacement activity as DNA polymerase to catalyze new chains synthesis under the condition of constant temperature. Then the targeted gene was amplificated efficiently.3We optimized the LAMP reaction system and reaction conditions. To make the results observed intuitive and accurate and finally achieve the quick and easy detection we added SYBR-Green I in the reaction system and combined with the color reaction.4We applied Standard strains of salmonella, non salmonella strains and Sample isolates strains of salmonella to detect the specificity and sensitivity of this method. We detected the salmonella in meat, eggs, milk, aquatic products in totally100samples by phenotypic detection method and color culture medium method synchronously to test the practicability of the method. Results:1. We established rapid detection of salmonella by LAMP method. We found the best reaction condition was that reaction volume25ul, best expansion temperature63℃and best reaction time60min.2. The minimum concentration of salmonella DNA and Salmonella bacteria liquid of this method was1fg/ml and10cfu/ml respectively. Positive rate of salmonella strains and negative rate of non salmonella strain were all100%.3. The consistent rate of LAMP and color culture medium method a type of phenotypic detection was100%in the detection of100samples.Conclusions:This study established the salmonella LAMP detection method with stn gene as target gene and applied to the detection of Standard strains of salmonella and samples may be contaminated by salmonella. The characters of this method was high sensitivity, high specificity, simple and rapid, without expensive instrument and equipment, simple operation, easy to observe the results and not easy to appear the DNA contamination etc. It really achieved the purpose of rapid detection of salmonella and was a suitable and rapid method for port inspection and quarantine departments at the grass-roots level on the detection of salmonella.