Establishment Of Visual Loop-mediated Isothermal Amplification(LAMP) For Detection Of Neo In Genetically Modified Pigs

With the continuous development of the transgenic technology, many genetically modified(GM) animals have come out, such as GM mice, fishes, rabbits, pigs, monkeys, cows, chickens.GM animals are widely used in medicine, health, agriculture and biological materials, and manyother fields, therefore, the security issue are also increasingly prominent.Nowadays, selectable drug-resistant genes are inevitably used in most of GM animals, such asneomycin phosphotransferase (neo), puromycin acetyltransferase (puro), hygromycin Bphosphotransferase enzyme (hph), xanthine/guanine phosphoribosyl transferase (gpt),hypoxanthine phosphoribosyl transferase (Hprt), diphtheria toxin (DT), thymidine kinase (tk) andthe like. Therefore, the detection of resistant marker genes in GM animals and their standards forbio-safety assessment are very necessary. There are many measures for detection of exogenousgene and its expression in GMO, including PCR, Southern blot, gene chips, ELISA, Western blot,protein chip. More stringent technical requirements and relatively expensive equipments orreagents are needed, which are not suitable for the detection in the grassroots.Loop-mediated isothermal amplification (LAMP) is a new novel nucleic acid amplificationmethod, which has been invited in2000by Notimi T, from Eiken Chemical Co. Ltd. in Japan. It hasmany advantages, specificity, high sensitivity, simple and time-saving. The operator does notrequire a rigorous professional training and expensive instrumentations, especially in grassroots.LAMP can meet the requirements of rapid detection site and has a wide spread of applicationprospects.In this study, a visual and rapid detection method of exogenous marker gene in GM animalswas established, based on loop-mediated isothermal amplification (LAMP). Successfully wescreened out a set of specific LAMP primers, established and optimized the LAMP reaction system,which could be completed within a range of50-60min; LAMP method has the sensitivity to2pg/μl,three orders of magnitude higher than PCR for detection of neo in GM pigs; LAMP methodreached98%consistency with PCR when detected50GM pigs. The results of this study canprovide a new method for the detection of resistance gene in GM animals.