Study On Detection Of Salmonella In Meat By Real-time Fluorescence Loop-mediated Isothermal Amplification Method

In public health, Salmonellosis is one of the most significant zoonosis, Because of theFoodborne Disease. In every year there are at least50%of foodborne illness is caused bysalmonella.but the detection method of salmonella is to long time, specificity andsensitivity is not high.This is no longer adapt to the development of The Times.In today’srapid development,Set up a high sensitivity, high specificity and a short times method isnecessary.Real-Time Fluorescence Loop-mediated isothermal amplification (LAMP) is a newmicrobiological method.The Fluorescence was added to the LAMP reaction system, usingthe accumulation of fluorescence signal as the reaction time monitoring the progress of thereaction in LAMP,and through the analysis software to test the LAMP reaction analysistechnology.(LAMP) has been used frequently in a lot of areas including the rapid detectionof pathogens.In this study by optimizing the system of Mg2+,dNTPs,Bet,BstDNApolymerase,concentration of template and the temperature of the reaction,the propotion ofinside and outside Primer’s to determine the optimal Real-Time FluorescenceLoop-mediated isothermal amplification.The reaction mixture consisted of2.5μL10×Bstbuffer, concentration of Mg2+is1μL, Concentration of dNTP is5μL,concentration of BstDNA polymerase is1μL, concentration of betaine is1μL,concentration of template is1.5μL, concentration of SYBR is0.5μL, each of FIP/BIP primer2.5μL, each of F3/B3primer0.5μL,6.5μL of double-distilled water. The reaction was run under61℃for1h.this study in order to checking the specificity of real-time fluorescent LAMP primers,selected the common pathogenic bacteria in response.The results show that Salmonella waspositive,the others strains were showed negative.This study compares the real-time fluorescent LAMP method and the PCR sensitivityto detect the pure bacteria and detection limit of artificial contaminated meat. the resultsshow that: the sensitivity of Real-Time Fluorescence LAMP detection of Salmonella stainswas5.6×101CFU/mL,but the PCR detection of Salmonella stains was5.6×102CFU/mL.real-time fluorescent LAMP method and PCR method detection limit ofsalmonella in artificially contaminated meat was5.6×104CFU/mLand5.6×105CFU/mL.This study establish the Real-Time Fluorescence LAMP assay of detection ofSalmonella successfully, which is high specifical and sensitive, also save time for thedetection of pathogens in food. So it provides the technology platform and technical support for the general detection organizations to promote this technology.