Study On A Loop Mediated Isothermal Amplification (LAMP) For The Detection Of Enterobacter Sakazakii In Infant Formula

In 2002, the International Commission for Microbiological Specifications for Foods (ICMSF) ranked the organism as'Severe hazard for restricted populations, life threatening or substantial chronic sequelae or long duration'. Enterobacter sakazakii has been detected in other types of food, but only powdered infant formul has been linked to outbreaks of disease. Because a few cells of E. sakazakii (<3CFU/100g)in powdered infant formula can cause severe impact on health in short time, the development of a rapid, sensitive and specific method for early detection of this bacterium in clinical research and food protection is urgent.At present, the FDA-recommended methods for isolation and identification of E. sakazakii from dehydrated powdered infant formula.These methods are time-consuming and labor intensive and also are lower sensitivity.The sensitivity and specificity of immunological Methods are unsatisfactory. Recently, a number of new molecular biological methods for rapid detection of bacteria have been reported, including polymerase chain reaction (PCR). The PCR methods provide powerful tools for rapid, specific, and sensitive detection of food-borne pathogens and are considered reliable alternatives for traditional bacteriological methods. However, owing to the expensive systems required and complicate electrophoretic analysis, this application is still not very common in laboratories.A loop-mediated isothermal amplification (LAMP) technology, which can provide a sensitive, specific, simple and cost-effective test for the rapid detection of Enterobacter sakazakii in powdered infant formula, was conducted in this assay. It can popularize and promote in common laboratories.We used the 16S–23S rRNA internal transcribed spacer (ITS) sequence of Enterobacter sakazakii (ATCC29544) as target sequences and used Primer Explorer software, version 3(https://primerexplorer.jp/lamp3.0.0/index.html), to design LAMP primers. The reaction conditions were optimized including temperature, time and Mg2+ concentration etc. The LAMP mixture was made in 25μL of reaction mixture containing a 1.2μM concentration of each inner primer (FIP and BIP), a 0.2μM concentration of each outer primer (F3 and B3), a 0.6μM concentration of the loop primer(LF and LB), 2.5 mM each deoxynucleoside triphosphate, 1.6 M betaine, 4 mM MgSO4, 10×Bst DNA polymerase reaction buffer, 8U of the Bst DNA polymerase large fragment, 3μL isolated DNA templates and sterilized double-distilled water. The mixture was incubated at 64℃for 20min and then heated to 80℃for 10min to terminate the reaction. We can obtain the results of detection when the white precipitate was observed by visual examination.For an assessment of the sensitivity and detection limit of artificial contamination of LAMP, PCR with the two outer primers (F3 and B3) of LAMP with two loop primers was conducted. We extracted DNA using the same DNA kit method. The detection limit of pure bacterial culture was 0.101CFU/mL and the detection limit of artificially contaminated powdered infant formula was 1.1CFU/g with improved LAMP detection for an hour. In contrast, the detection limit of pure bacterial culture was 101CFU/mL and the detection limit of artificially contaminated powdered infant formula was 1100CFU/g with PCR detection for three hours. Thus, the sensitivity of the improved LAMP of pure bacterial culture and artificially contaminated powdered infant formula were both 1000 times as high as that of PCR.In this study, comparison of the sensitivity for detection Enterobacter sakazakii and detection limit of artificial contamination of the improved LAMP and the general LAMP,as well as the improved LAMP with loop primers and without loop primers,the results show that improved LAMP sensitivity was the general LAMP and without loop primers of improved LAMP 10 times.The effect of six methods of extracting DNA from Enterobacter sakazakii in infant formula were compared, LAMP, the method of extracting DNA, no strict requirements, inhibiting factors are also less than the PCR. In this study, real detection was made. The result indicates: the sensitivity of the method is 100%, the specificity is 98.15%, and the coincidence rate is 98.25%.The result indicates: LAMP has the potential to replace PCR because of its simplicity, rapidity, specificity and cost-effectiveness. In our study, we had developed a new and rapid molecular biological method for detection of E. sakazakii in food sample. For the rapid detection of foodborne pathogens build a technology platform.