Construction Of Loop-mediated Isothermal Amplification For Rapid Detection Of African Swine Fever Virus

African swine fever (ASF) is a high acute mortality disease caused by the African swine fever virus (African swine fever virus, ASFV), which is caused potent, contiguous and infectious. The mortality of the animals reaches to 100% after the infection of the virulent strain. There is no effective preventive vaccine for African swine fever at present. The global pig industry is influenced by this disease, and it caused a huge threaten and economic losses. Although there is no occurrence of African swine fever in our country in the moment, with the development of the increasing and frequent export trade, as well as the movement of person, the possibility of the introduction of this disease goes into China is also increased. In order to detect African swine fever in time, prevent the disease from entering China, thus establishing a rapid detection method for the laboratory diagnosis of African swine fever virus is very important. This study is on the base of the molecular biology, taking use of the fast and simple features of the loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP), then establish a rapid detection of African swine fever virus LAMP methods.In this experiment, through the research of the molecular biology of African swine fever, the synthetic plasmid that containing the K205R gene of African swine fever virus is synthesized, this synthetic plasmid is used as the required template, according to the K205R gene of African swine fever sequences that have been logged in NCBI. Through online software, according to the design principles of the LAMP primer, designing a specific primer for the K205R gene sequences of African swine fever virus, after the optimization of the reactive conditions, successfully establishing the methods of the LAMP of African swine fever virus. Through the identification and the analysis of the reactive products, as well as the comparison of sensitivity tests and conventional PCR methods, the results showed that the LAMP detection methods of African swine fever can be detected below the 10 copies of the African swine fever DNA, it has a higher sensitivity and better specificity and also better stability. This method is simple and rapid, the reaction can be completed in 45 minutes with the watering bath that has a constant temperature of 63℃.The reactive products can be analyzed by agarose gel electrophoresis, besides, a visual inspection is also available, may be positive reaction tube became turbid under natural light, and it appears the green fluorescence after adding a fluorescent dye, under the UV light shining. Because the features of the LAMP products that can be detected by vision, coupled with the simple reactive conditions, and it does not require sophisticated instruments and equipment. LAMP has been widely used in a variety of disease detection,it is a suitable laboratory and on-site rapid detection of methods. The establishment of this experimental methods provides our country with a reference that can prevent the epidemic infection of African swine fever in our country.