The Development Of Rapid Assay For African Swine Fever Virus By Loop Mediated Isothermal Amplification Method (LAMP)

African swine fever (ASF) which was caused by African swine fever virus (ASFV) had a lethality rate of 100% and was declared to be one of the"list A"diseases by OIE. There was no effective therapy up to now for the special immune evade mechanism of ASFV. Although no ASF has been reported in China; with the fast development of international communication, threaten of ASF also increases. Developing a fast; sensitive; efficient and economic detection method for ASF is urgent for China's bio-security. Loop mediated isothermal amplification method (LAMP) has been wildly used in pathogen detection for its inimitable design concept. As there was no ASFV-LAMP method has been reported yet, this research has not only application values but also scientific values.The highly conserved P72 gene of ASFV is a good target for rapid assay by nucleic acid techniques. In this research, part of the conserved area at the C-term of P72 gene was picked by bioinformatics method to design LAMP primers. Four sets of primers were designed and validated by using software depending on some important parameters including: GC content, 5'-,3'-term stability and secondary structures, et al. Restriction sites were also designed in each amplification product for later identification by enzyme digest. Results of amplification by using conserved parameters show that the fourth set of primers (4FIP, 4BIP, 4F3, 4B3) has the best efficiency. And reliability has been proved by enzyme digest.In the following step, several important parameters of LAMP were optimized including temperature, Mg2+, outer and inner primers, and betaine concentration. Mg2+ and inner primers were found to influent most on amplification efficiency after research. Best result was got with a Mg2+ concentration of 8mM and inner primer concentration of 1.6~2.4μM. Optimizing temperature, outer primers and betaine can also improve the amplification efficiency but have a limit when the value comes to a certain level.Based on the optimized primers, the ASFV-LAMP method was developed. A set of time cost, sensitivity, specificity and reliability tests have been done with a comparison of OIE standard ASFV-PCR method. It has been proved in time test that only 30 min would be taken by ASFV-LAMP to get positive result, much less than PCR. In sensitivity test, the detection limit was found to be 5 copies of template which was validated by using serial diluted templates. The sensitivity of ASFV-LAMP is 100 fold of the OIE standard PCR method; which can still be improved by adding loop primers. Specific experiment showed that there were no non-specific amplification occurs when using porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and Porcine Pseudorabies Virus (PRV) genomes as templates. In repetitive and stability tests, experiments with 8 batches of templates and 8 experiments with the same templates have been done. Stable outcomes guaranteed the reliability of ASFV-LAMP method.To sum up, a rapid, sensitive, efficient and reliable ASFV-LAMP method have been developed in this research, which is a firm foundation of the following development of ASFV-LAMP rapid assay kit with high application value.