Loop-mediated Isothermal Amplification For Rapid Detection Of O157:H7Enterohemorrhagic E. Coli

Escherichia coli O157:H7is a widespread pathogenic germ which can infect animals and human beings, it is widely distributed in nature, which is the main pathogens causing diarrhea and is a great health hazard to human and animals. In a word, there are many cases, such as alimentary toxicosis, which caused by Escherichia coli O157:H7is now a very serious public health and safety issue at the home and abroad. With the vigorous development of waterfowl breeding industry, and the disease caused by E.coli has already existed in my country, but the research of E.coli O157:H7is quite rare. Therefore, the establishment of a fast and efficient detection of E. coli0157:H7is particularly important, which was used to protect the development of breeding industry in waterfowl.Currently, the commonly used methods for isolation and identification of E. coli O157:H7involved routine cultivation method, ELISA, and PCR. Traditional method which was used to detect the E.coli O157:H7is time-consuming, complex and lower sentitivity. Immunological method was not so satisfied in the sensitivity and specificity. Although the PCR methods had the advantages of rapid, sensitive and accurate for detection, but due to the higher demand for the equipments and higher quality requirements for the operating personnel, this technology is still not very common in the grassroots areas.This experiment aimed the rfbE gene sequence of E.coli O157:H7(Genbank S83460) at the target sequences, and the LAMP primers were designed by the help of Primer Explorer software. The reaction conditions were optimized, such as the primer Proportion, tempreture, and the amount of dNTPs, Mg2+, and Bst DNA polymerase etc. The reaction system of LAMP was identified as25μL of mixture. Amplification reaction program:incubated at water bath62℃,30minutes, after that neated to80℃for3minutes to terminate the reaction. Finally, centrifuging the LAMP reaction tubes for a few seconds and judging whether the reaction happened or not with the help of the observation of cloudy white precipitate, or adding product SYBR Green I fluorescer dye in order to judge the results by observation of the colour Change, or to take5μL product mixed1μL6x lodding buffer in2%agarose gel electrophoresis and finally, using the gel imaging system to observe whether there were ladder-shaped strips to judge the results.Compared with the PCR detection method, this research made an assessment of the sensitivity of this LAMP method, and using the outer primer (F3and B3) as the Forward primer and reverse primer of PCR reaction.The study showed that:the detective limitation of pure bacterial culture of LAMP method was2.20x102CFU/mL, and the limitation of PCR method was2.20x104CFU/mL, therefore, we can see the ensitivity of LAMP reaction was100times of the PCR reaction. In this study, we used the kit method to extract DNA. From the initial sample processing to finish the report, compared with the PCR reaction needed for4hours, the LAMP reaction only needed2hours. In the meantime, in this research, this LAMP method had a high specificity for the detection of E. coli0157:H7by amplifying a fragment of rfbE gene rather than other seven pathogenic bacterias (E.coli O157:H7, Bacillus subtil, Bifidobucterium pullorum, Riemerella anatipestifer, Staphylococcus, Pasteurella, Salmonella, E. coli046)From August2010to January2012,251strains of E.coli were collected from, Beijing, Guangdong, Guangxi, Sicuan, Jiangsu and other areas, the detective results of LAMP assay showed thatonly one srain of E.coli was detected as E. coli0157:H7, so the positive rate was only0.4%.This research showed that the LAMP assay which were established to detecte for E.coli O157:H7had the advantages of high sensitivity, strong specificity, time-consuming and the operating program was also simple, which provided newideas for the detection of E.coli O157:H7, and this method also did not need costly instrument, if the problem of false positive can be avoided effectively, this method will greatly promote the quarantine work in the grassroots areas.