牛乳及饲料中三聚氰胺的检测方法研究食品加工与安全专业论文.docxVIP

Real-Time Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Diagnosis ofShigella in milk Name：Shen LU Major：Food processing and security Supervisor：Professor Zhang Wei Abstract Shigella is representative of the gram—positive bacteria．On the hand，Shigella call cause pyogenic inflammation，on the other hand，after contaminate food Shigella produce Staphylococcal enterotoxin food poisoning．It is recognized one of the major of human food—borne diseases．The pollution probability of meat，eggs，milk and their products is bigger．In recent years，a growing number of Shigella food poisoning has become worldwide health problem．At present，the detection of Shigella takes long time，is not sensitive enough，and is not for real—time monitoring of reaction process，thus to create a new kind of rapid and sensitive detection method of Shigellais necessary． The real-time loop·mediated isothermal amplification we used in this study is the improvement of loop-mediated isothermal amplification．Put fluorescent dye SYBR Green I into LAMP system，SYBR Green I combine to double．stranded DNA．and the fluorescence signal will enhance thousands of time．Put the reaction system into real-time fluorescence detecting instrument for reaction，through the corresponding software observed reaction process．This method terminate the experiment at any time to control the reaction process． Based on the gene sequences of S in GenBank，according to Primer design principle， this study online Primer design software Primer V4 designed and selected pair of specific primers to match the gene sequences of s．auregs．By adjusting the reaction conditions of this study, established the following reaction system： I．6UM FIP and BIP,0．4pM F3 and B3，2mM MgS04，0．35mM dNTPs，2．5pl 1 0xBst DNA polymerase buffer,12U Bst DNA polymerase，O．51al DNA template，O．5雎l I：350 dilution SYBR Greenl fluorescent dye，6．5 1al sterilized distilled water．Set the reaction temperature is 6 1℃，reaction time is 90 min，by using fluores