Study On Simultaneous Detection Of Salmonella Spp. And Shigella Spp. By Loop-mediated Isothermal Amplification

Microbial contamination is one of the main factors which affect food hygiene and safety,among them bacterial food poisoning caused the largest number of poisoning. Rapid detection of pathogenic bacteria in food is a timely and effective prevention of spread of pathogenic bacteria and an important prerequisite for food poisoning.Presently, detection of pathogenic bacteria mainly rely on conventional method of bacteriological culture,normally takes 4d～7d,which are cumbersome and time-consuming.Loop-mediated isothermal amplification(LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity,efficiency and rapidity under isothermal conditions.This method employs a DNA polymerase with strand displacement activity and a set of four specially designed primers.LAMP can amplify a few copies of DNA to 10⁹ in less than an hour.The final products are stem-loop DNA with several inverted repeats of the target and cauliflower-like structures with multiple loops.A positive reaction would be shown as a ladder-like pattern in gel electrophoresis analysis.Because of its high specificity,sensitivity,rapidity,accuracy and simple to operate,LAMP method will be widely applied to research on nucleic acid,clinical diagnosis of infectious diseases and detection of genetically modified organisms etc.Two sets of primers were designed according to the known sequences of Salmonella spp.invA gene and Shigella spp.ipaH gene.Using LAMP method to amplify genome DNA,rapid detection method was established to detect Salmonella spp.and Shigella spp. simultaneously,and was used in milk samples artificially contaminated with pathogens. The results are as follows:1.Results of primer design for double LAMP and specificity of the primers(1) According to the Salmonella spp.invA gene and Shigella spp.ipaH gene.,primers were designed using PrimerExplorer 5.0 and were analyzed by Oligo 6.0 to avoid the formation of steady dimer.Moreover the primers and amplification fragments were analyzed by BLAST to avoid high homology and complementarity through GeneBank, two sets of primers were obtained invA-F3:5'-GCGATAATATGGGGCGGAAT; invA-B3:5'-CGCCTTTGCTGGTTTTAGGT; invA-FIP:5'-TCCCGGCAGAGTTCCCATTGAAATCATGACGCAGCTGTTGAA; invA-BIP:5'-TTCCCGCTGCCGGTATTTGTTGCTACGTTTTGCTTCACGGA; invA-LB:5'-GCCGTAACAACCAATACAAATGG; invA-LF:5'-TATTCGGTGGTTTTAAGCGTACTC; ipaH-F3:5'-GCCTTTCCGATACCGTCTCT; ipaH-B3:5'-TGATGGACCAGGAGGGTT; ipaH-FIP:5'-TCCGCAGAGGCACTGAGTTTTTCACGCAATACCTCCGGATTC; ipaH-BIP:5'-TCGACAGCAGTCTTTCGCTGTTCCGGAGATTGTTCCATGTGA; ipaH-LB:5'-TGCAGCGACCTGTTCACG; ipaH-LF:5'-CTGCTGATGCCACTGAGAGC.(2) The LAMP amplification conditions of Salmonella spp.were optimized and the specificity test of primers was developed.The results showed that the optimized reaction conditions of Salmonella spp.followed as the concentration of outer primer invA-F3 and invA-B3 5 pmol/L,inner primers invA-FIP and invA-BIP 40 pmol/L,loop primers invA-LB and invA-LF 20 pmol/L,6 mmol/L Mg²⁺,0.8 mmol/L dNTP,incubated for 45 min at 63℃and heated for 2 min at 80℃.Primers specificity test showed that Salmonella spp.primers were positive to all tested Salmonella spp.and negative to all other bacteria,which indicated the primers were specific.Under the condition,the detection limit for Salmonella spp.DNA template was 10 fg/tube.(3) The LAMP amplification conditions of Shigella spp.were optimized and the primers specificity test was developed.The results showed that the optimized reaction conditions of Shigella spp.followed as the concentration of outer primer invA-F3 and invA-B3 5 pmol/L,inner primers invA-FIP and invA-BIP 40 pmol/L,loop primers invA-LB and invA-LF 20 pmol/L,8 mmol/L Mg²⁺,1.0 mmol/L dNTP,incubated for 45 min at 63℃and heat for 2 min at 80℃.Primers specificity test showed that Shigella spp.primers were positive to all tested Shigella spp.and negative to all other bacteria,which indicated the primers were specific.Under the condition,the detection limit for Shigella spp.DNA template was 1 fg/tube.2.Development of double LAMP assay for Salmonella spp.and Shigella spp.The optimized reaction conditions followed as the concentration of outer primers invA-F3,invA-B3,invA-F3 and invA-B3 5 pmol/L,inner primers invA-FIP,invA-BIP, invA-FIP and invA-BIP 40 pmol/L,loop primers invA-LB,invA-LF,invA-LB and invA-LF 20 pmol/L,8 mmol/L Mg²⁺,1.0 mmol/L dNTP,incubated for 45 min at 63℃and heated for 2 min at 80℃.Under the condition,the detection limits for DNA template wase 10 fg/tube.But PCR can only detect 1 pg/tube from DNA samples.Milk samples which inoculated Salmonella spp.and Shigella spp.were detected.The results showed that the sensitivity of LAMP could reach 5 cfu/10 mL samples,which indicated that the method was with high sensitivity.Double LAMP assay for simultaneous detection of Salmonella spp.and Shigella spp. have been established in the study and applied to milk samples contaminated pathogens artificially.This method can be the supplement of traditional method and applied to food microorganism detection,which can improve the speed,accuracy and sensitivity.