| Egg Drop Syndrome (EDS-76) was a virulent infectious disease caused by EDSV with the main characteristics of laying shelless or soft shelled eggs and decreasing egg laying. Since the first reported of Van Eck in 1976, many virus strain of EDSV was isolated around the world. The virulence between strains was large differences, the host ranges widely and the virus can be reproduced in the body long. There was no cross reaction between fowl adenovirusⅠand fowl adenovirusⅡand have Unique antigenicity and biological properties. Its occurrence and epidemic in the world caused enormous economy lose to poultry industry, so it is very important to understand of the genome structure characteristics and to establish a rapid and specific diagnosis method.According to the published genome sequences of egg drop syndrome virus(EDSV-76) Duck adenovirus A on GenBank (AC000004), twenty-two pairs of primers were designed by software Premier 5.0 and the complete genome of egg drop syndrome virus NE4 strain which was amplified in the duck embryonated eggs was amplified by PCR. Splicing each fragment by DNAStar software and analyse the homology of EDSV proteins with other adenoviruses which GenBank have registered. Hexon protein was used to build the Phylogenetic tree. The results showed that the genome sequence of NE4 strain was 33214bp in length and the GC content is 43%. Compared with the genome of International standard strain AV-127, the nucleotide similarity was 99.6%. Insertions or deletions of bases mainly located in the noncoding region. There was a base C inserted in one-tenth from the C-terminal in the 100K protein coding region,the translation was terminated in advance. So the ORF encoding 696 amino acids while the AV-127 strain encoding 709 amino acids. The N terminal is expected to be function region. Protein homology analysis showed that the main proteins have a high homology with OAV(Ovine adenovirus D) , BAV(Bovine adenovirus D) and SAV(Snake adenovirus),but low homology with CELO. This indicated that there was a big difference between NE4 strain and fowl adenovirusⅠin serology and gene structure. Phylogenetic analysis also indicated this feature. All of this is helpful for further studying EDSV genome structure, its homology with other adenovirus relations, and providing a clear molecular biology background for EDSV as a adenovirus vector.At present, the main methods to detect EDSV are Hemagglutination inhibition test, Agar diffusion test, Enzyme-linked immunosorbent assay test, PCR and Immunogold detection. Following the development of molecular biotechnology, some new technologies, such as Loop-mediated isothermal amplification method (LAMP), are developed in the recent years and supply new skills for the early and rapid examination of EDSV.In order to develop an efficient, rapid and highly specific loop-mediated isothermal gene amplification (LAMP) assay to detect egg drop syndrome virus (EDSV), three pairs of primers were designed according to hexon conservative region of EDSV. The LAMP was performed using Bst DNA polymerase large fragment under the effect of isothermal amplification. The LAMP reaction system was optimized and the sensitivity and specificity was tested, The result showed that the products amplified by LAMP from EDSV were distinctively ladder-like band in agarose gel electrophoresis, and green fluorescence should be seen in colorable reaction. The sensitivity of LAMP assay developed in this study was 60 copies.μL-1. It was 10 times higher than that of PCR. It have no cross reaction with NDV,IBDV,IBV and CELO . The LAMP assay was more simple and convenient, which can be used economically and effectively in both laboratorial and pen-side testing. |
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