A Rapid And Sensitive Method Using For Detection Of Escherichia Coli. O157:H7

Diarrhea caused by E. coli O157:H7 is a serious medical disease in recent years. Since E. coli O157:H7 was first identified as a cause of illness in 1982, more than forty countries have this disease outbreak in different degree and with high mortality. World Health Organization identified E. coli 0157:H7 is a new foodborne disease pathogens. Now, there are many kinds of method using for detection of E. coli 0157: H7, but most of these have some problems such as need a long time or need some special equipments or cannot reach a high accuracy. Therefore, it is very important to search a convenient and sensitive way to detect E. coli O157:H7 to prevent diseases caused by this bacteria.First, using the immunomagnetic nanoparticles that was coated with the antibody of E. coli O157:H7 to enrich the bacteria to increase the concentration of the E. coli O157:H7 in the sample. Choose two sizes of the immunomagnetic bead that are 1μm and 200nm and to research the influence of the size of the magnetic bead with the enrichment efficiency. And then choose the best reaction conditions. The results show, using 1.2μL of 200nm magnetic bead to enrich 1mL of the E. coli 0157:H7 with a low concentration is proper.We construct a simple and rapid bacteria genomic DNA purification method, named magnetic DNA purification method, according to the principle that polynucleotides can bind reversibly and non-specifically to the surface of carboxylic acid modified magnetic microparticles, under a high concentration of salt, but proteins, monosaccharides, polysaccharides and lipids can not.This method can enrich and purify the genomic DNA of E. coli O157:H7 efficiently within 7 minute. This method is simple, time-saving and independent on the centrifuge and organic solvent.Finally, LMAP method was used for the rapid detection of DNA in the E. coli 0157: H7 and explore the optimum conditions. The results show this method can detect E. coli 0157:H7 that is 30CFU/mL. The LAMP reaction requires four or six primers, which are based on six or eight distinct regions of the target DNA, and using Bst E which has polymerase chain replacement activity. LAMP is easy to perform and can amplify nucleic acid at isothermal conditions. Compared with PCR, it is quicker more specific and does not need any special equipment. LAMP is ten times more sensitive than PCR.This paper combined immunomagnetic beads technique and magnetic bead to extract DNA and used LAMP for detection of E. coli 0157:H7. The whole process dose not require any special equipment, and can be finished within70 minutes. This is a good method for application. And we offer an excellent way for detection of E. coli 0157: H7 and to make automatic detection of E. coli.0157:H7 much quicker.