Using Real-time Fluorescence Loop-mediated Isothermal Amplification To Detect Bacillus Cereus In Milk

In recent years, with the development of China’s food industry, the number and types of food have been growing by leaps and bounds,but food poisoning caused by foodborne pathogens continue to occur, improving the current microbiological testing method for detecting a long time, tedious steps, low sensitivity and some defects, to meet the rapid, accurate, simple to detect pathogens in food has become a matter of concern. Bacillus cereus is widely present in the surrounding environment, and can easily contaminate food, especially high protein and high carbohydrate food, it can easily cause food poisoning,usually is diarrhea and vomiting. Currently, for Bacillus cereus detection of commonly used traditional national standard method, reliable this method, although the test results, but the test method procedures range detection long the result of time, and the new rapid detection method, such as immunization high school method, polymerase chain reaction, biosensor technology, etc. are more or less testing costs, unreliable, or other factors on the higher quality requirements of professional operators, these methods do not result in a promotion play-scale use.Notomi T. et al., Developed a loop-mediated isothermal amplification(Loop-mediated isothermal amplification, referred to as the LAMP) in 2000, four kinds of specific primers designed for the six regions of the target genes, the use of having a strand displacement activity DNA polymerase enzymes(Bst DNA poly-merase), efficient amplification reaction at a constant temperature, and dozens of minutes to complete, and its amplification products either by electrophoresis to detect, or by adding dye SYBY GreenⅠdetermine with the naked eye,or magnesium pyrophosphate turbidity detection. Real-time fluorescence loop-mediated isothermal amplification reaction is a new detection technologies developed on the basis of LAMP. LAMP reaction based on merit and improved LAMP defects.This study is based on LAMP, adding a fluorescent dye in the reaction system, using real-time fluorescence monitor(ESE-Quant Tube Scanner) for real-time monitoring, observation directly on the computer.In this study, for hbl A gene of Bacillus cereus, the use of online primer design software designed four primers.Bst enzyme dosage, d NTP concentration, magnesium ion concentration, and some amplification conditions were optimized. Using real-time fluorescence monitor the reaction temperature at 63 ℃ 80 min to detection of Bacillus cereus. Final optimized reaction system: outer primers F3 and B3 each 0.5μL, inner primers BIP and FIP 2.5μL, Mg SO4 final concentration of 1.0 m M,0.3m M d NTPs, 10 × Theromo Pol Reaction Buffer 2.5μL, Bst DNA polymerase(8000U) l.5μL, DNA template 2μL, betaine 0.5μL, fluorescent dyes 0.5μL, dd H2 O make up the volume to 25μL. Amplification reaction conditions: 63 ℃ reaction 80 min.In order to verify the specificity of the method, this study use four Bacillus cereus and 17 un-Bacillus cereus to detection by real-time fluorescence LAMP, the results showed that four of Bacillus cereus showed positive, while the other 17 un-Bacillus cereus showed were negative, indicating the high specificity of the test method.When testing sensitivity of real-time fluorescence LAMP,the result shows that this method of Bacillus cereus sensitivity up 8.2CFU / m L, 10 times higher sensitivity than conventional LAMP. Artificially contaminated milk of real-time fluorescence LAMP detection limit of 8.2CFU / m L.Studies have shown that: This study successfully established a real-time fluorescence LAMP detection of Bacillus cereus technology that can achieve fast, simple, high sensitivity and specificity to detect Bacillus cereus, presented to the Bacillus cereus in food testing new ideas.