Construction Of Chicken Infectious Anemia Virus Infectiousness Clone And The Establishment Of LAMP Examination Method

Chicken anemia virus (CAV) is the pathogen of Chicken infectious anemia(CIA). CAV belongs to Circoviridae.CAV genome encodes three proteins:VP1 (51.6kD), VP2(24kD) and VP3(13.6kD). VP3 (Apoptin) can induce cellular apoptosis.The nucleotide at site 487nt of CAV genome was changed from T to C and the nucleotide at site 526nt was changed from C to T through gene mutation technology.As a result,the promotor of VP3 was shifted to the position where the original 14th amino acid was 39 bases were absent and therefore the expression of VP3 will be incomplete. However,the expression of VP2 will not be interfered as the amino acids in related region have not been changed.Further more,incomplete EcoR I recognition sequence (5'-GAATTC-3')at CAV non-coding area was modified into complete EcoR I recognition sequence (5'-GAATTC-3')through site-direct PCR mutation, which is required for CAV genome re-ligation in vitro. The re-ligated mutant CAV has been transfected into the host cell line (MDDC-MSB1). Futher study will be focused on the impact of the mutant to the viral pathogenicity and hopefully this approach can lead to the development of a non-virulent or attenuated CAV vaccine.Loop-mediated isothermal amplification (LAMP) technique is a novel nucleic acid amplification method which relies on autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment.It is an idothermal reaction uses a single temperature and the result can be directly judged through the colormetric detection of nucleic acid dye or judged from the foumed pellet. In this research, six pairs of primers were designed based on the CAV genomic DNA sequence, and LAMP detection method of CAV Cux-1 was built using the primers. Most of the componemts and conditions of the reaction including the content of Mg2+ in LAMP Buffer, the annealing temperature and the extension time were optimized and examed by agarose gel electrophoresis and direct dyeing observation with naked eyes. Additionally, the reaction specificity and its detection limit was 10 coyies a convenient and rapid method to diagnose CIA. Field experiment proved that the LAMP method for CIA detection was a convenient and rapid method, and its sensitivity could meet the need of CAV diagnosis and inspection.