The Establishment Of A Rapid, Convenient And Accurate Method Based On The 5 Exogenous Nucleicacid Of Transgenic Pig, Cattle And Goat

As a hot topic, the security managements to transgenic animals and their products play an important role since the marketization of such animals are inevitable in our society. Detection is crucial for safety managements. There are two main ways for transgenic animal detections, the nucleic acid and protein levels of the exogenous genes. Since the nucleic acid operations are more convenient than the protein levels, the construction of a rapid, convenient and accurate method based on the nucleic acid level is crucial and urgent for the transgenic detections.Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method. It is characterized by the use of 4 different specifically designed primers to recognize 6 distinct regions on the target gene and the reaction proceeds at a constant temperature about (65℃) in less than 60minutes using a DNA polymerase with strand displace activity. When adding SYBR GreenⅠinto the LAMP products, the detection can be finished by naked eyes. Since LAMP has the advantages of reaction simplicity and higher amplification efficiency, we chose LAMP to setup a simple, rapid, sensitive and accurate detecting method for transgenic animals at nucleic acid level. The results are as follows:1. Established a method by Loop-mediated isothermal amplification (LAMP) for the detection of exogenous gene from LYZ transgene-induced cattle, LTF transgene-induced cattle, LALBA transgene-induced cattle, FAT1 transgene-induced pig, HBsAg transgene-induced goat.2. Develop a visualization inspection for LAMP products, which could confirm the existence of target gene without agarose gel electrophoresis.3. For further confirmation, the LAMP amplified products were digested with restriction enzyme and detected by agarose gel electrophoresis.4. The detection limits of LYZ, LTF, LALBA, Fat-land HBsAg by LAMP methods were finished by comparing to PCR, and LAMP was found to be 10-100 fold more sensitive than PCR.5. After using the LAMP methods to detect the LYZ transgene-induced cattle, LTF transgene-induced cattle, LALBA transgene-induced cattle. FAT1 transgene-induced pig, HBsAg transgene-induced goat, the detectable rate were up to 100%.