Study On Loop-mediated Isothermal Amplification Assay For Detection Of Salmonella In Meat Products

Food safety emergencies frequently occurred in recent years.Salmonellosis is one of the most significant zoonosis,which is caused by salmonella belonging to intestinal bacteria branch,including bacterium leading to gastroenteritis,typhoid and paratyphoid. Human and animal infections can be a silent carrier state,or a lethal state manifesting clinical symptoms.It may increase the rate of mortality or death,or reduce the propagable productivity of animal.To ensure food safety and business,it is required to establish a rapid,sensitive salmonella testing method in food realm.A novel nucleic acid amplification method,termed loop-mediated isothermal amplification(LAMP),which amplifies DNA with high specificity,efficiency, andrapidity under isothermal conditions,may be a valuable tool for the rapid detection ofinfectious diseases.This method employs a DNA polymerase that have activity ofstrand displacement DNA synthesis and a set of four specially designed primers thatrecognize a total of six distinct sequences on the target DNA.LAMP can amplify afew copies of DNA to 10⁹ in less than an hour.The final products are stem-loop DNAwith several inverted repeats of the target and cauliflower-Iike structures with multipleloops.A positive reaction would be shown as a ladder-like pattern in a gelelectrophoresis analysis.Because of the advantage,the LAMP method will be widelyapplied to research of nucleic acid,clinical diagnosis of infectious diseases anddetection of genetically modif ed organisms etc.This study designed two pairs of gene-specific primers of conservative invA gene which have been reported by the salmonella,Mg²⁺concentration,dNTP concentration, betaine concentration,the temperature of the reaction and the propotion of FIP/BIP than F3/B3 are optimized to determine the optimal LAMP.The reaction mixture consisted of 2.5μL of 10×Bst buffer,4μL of dNTPs(1 mM each in solution),each of FIP/BIP primer 4μL(10μM stock solution),each of F3/B3 primer 0.5μL(10μM stock solution),the concentration of Mgcl2 is 2.5mmol/L,1μL Bst DNA polymerase(SU/μL of stock solution),4μL of betaine and 2μL of double-distilled water.The reaction was run under 62℃for 1h,the LAMP products were examined by electrophoresis with 2%agarose gel.The study compared the detection limit of the LAMP and PCR,the detection limit of the LAMP is 3cfu/μL,while 300 cfu/μL of PCR,so the detection limit of the LAMP is 100 times lower than PCR.The effect of seven methods of extracting DNA from Salmonella in meat were compared.An efficient extraction procedure was confirmed for extraction of Salmonella DNA from meat without enrichment.Salmonella DNA was directly extracted using FTA filter.The extracted DNA was suitable for LAMP detection. The LAMP method is high sensitivity and simple operation,Short time-consuming,this method is superior to other methods.48 samples were analyzed and the detection rate using the GB method was 85.4%, detection time was 5d.The detection rate of PCR amplification was 89.5%,100%for sensitivity and specificity of 83.8%,the according rate was 95.8%,the detection time was 6h.The detection rate of LAMP amplification was 91.6%,100%for sensitivity and specificity of 70.0%,the according rate was 93.8%,the detection time was 1.5h.This study establish the LAMP assay of detection of Salmonella successfully,which is high specifical and sensitive,also save time for the detection of pathogens in food.So it provides the technology platform and technical support for the general detection organizations to promote this technology.