Study On Real-time Fluorescence Loop Mediated Isothermal Amplification(RF-LAMP)for Detection Of Enterobacter Sakazakii In Powdered Infant Formula

Enterobacter sakazakii is a kind of gram-negative bacteria which is widespread innatural environment. E.sakazakii can not only exist in contaminated food and foodfactory, but also in almost all environments, including apartment. E. sakazakii candisease infant especially the newborn，such as meningitis，bacteremia and necrotizingenterocolitis，even serious sequelae or death. However, only existing in infant formulacan be associated with disease outbreaks. Therefore, to find out a sensitive, specific andfast method to detect Enterobacter sakazakii in infant formula is very important.Real-time fluorescent loop-mediated isothermal amplification (RF-LAMP)combines fluorescent dye with LAMP assay. The reaction principle is, when thefluorescent dye combines with double-stranded DNA, the fluorescence will be enhancedobviously. With the amount of reacting DNA increasing, the fluorescent signal isenhanced. Through the ESE-Quant tubescanner connected to the computer, the resultcould identify immediately. The tedious gel electrophoresis is not required to makeobservations. The method is simply operational, short time-consuming, real-timemonitoring, and the reaction results are intuitive.According to the16S rRNA gene sequences of E. sakazakii in GenBank(HQ880288.1), we used Primer Explorer software to design RF-LAMPprimers.Combination DNAMAN, Primer premier5.0screening the four specificRF-LAMP primers include F3/B3,FIP/BIP. The reaction conditions were optimizedincluding temperature, Mg2+and dNTPs concentration etc. The RF-LAMP reactionsystem (for example25uL) as follows：3.5μL of each inner primer (10μM FIP and BIP),0.5μL of each outer primer (10μM F3and B3),2.5mmol/L dNTPs2μL,50mM MgSO41μL,10×Bst DNA polymerase reaction buffer,8000U/ml Bst DNA polymerase0.8μL,2.0μL isolated DNA templates,0.5μL SYBR Green I and sterilized double-distilledwater.The most appropriate amplification reaction temperature is63℃, the amplify timeabout90min.But it can be stopped at any time according to reaction condition, thereaction, generally within60min. By restriction enzymes ACCIII enzyme amplificationproducts, measured enzyme fragment consistent with the expectations theory, verify thevalidity of the method. Three E. sakazakii strains and another twenty foodborne pathogenic bacteria strainswere used to test the specificity of detection. Among the23strains in test,3E. sakazakiistrains were identified and the other20strains were negative.To test the sensitivity and limit of detecting E.sakazakii and compared with ordinaryPCR method,the sensitivity and the limit of detecting E. sakazakii by RF-LAMP were47CFU/mL which was100times sensitive than ordinary PCR method and the testingtime was shortened greatly.This study conducts the real-time fluorescent loop mediated isothermalamplification detection assay of E.sakazakii in infant formula.It has the advantages ofhigh sensitivity, short time-consuming, high specificity and simple operation.The assaygreatly shorten the testing time,intuitively show test results and there must be a broadapplication prospect.