Development Of A Gene Chip Assay For The Detection Of Avian Leucosis Virus Subgroups B, E And J

Avian Leukosis (AL) has been harming poultry husbandry in recent years,and has become a the first big industry disease of egg chicken in our country. Since the common methods of laboratory waste time and energy,and the imported ELISA kit was expensive,a rapid, sensitive, accurate, high throughput test method for prevention and control of ALV is urgently required. In this study, a gene chip assay was developed to detect avian leucosis virus subgroups B, E and J based on the specific gene fragment amplified by multiplex PCR.By aligning multiple sequences of ALV subgroups B, E and J in GenBank, a conserved gene segment with multiple internal site variations was chosen as detection target. One forward primer for subgroups B, E and J, one reverse primer for subgroups B and E, and one reverse primer for subgroup J were designed to amplify the target gene in the three subgroups of ALV. The primers Cy3-labeled were used to amplify the target gene fragments from the template DNA by multiplex PCR. Five oligonucleotides from the internal variations of the target gene were synthesized and printed on the amino-modified slides as the probes. In order to make the probe length to be 35 mer, we add several bases T and make the probes amination in the 5’end of the probes, then spot the probes to the glass substrate. The template cDNA was obtained by either synthesizing based on the representative sequences of subgroups B, E and J of ALV in GenBank or reversely transcripting the extracted cDNA from ALV-infected DF-1 cell. The amplified products were hybridized to the oligonucleotide probes immobilized on the glass slides. Finally, Do some experiment for specificity and sensitivity of the gene chip, and test 50 clinical samples by the chip.After hybridization, the gene chips were scanned, and the hybridization pattern agreed perfectly with the known location of each probe from subgroups B, E and J. The detection sensitivity can be reached to 10~2 copies of gene. No cross-hybridization could be detected among 4 common avian viruses. Coincidence rate of the results for clinical samples testing of gene chip and ELISA was very high. The results demonstrated that the gene chip assay was an effective way to distinguish ALV subgroups B, E and J simultaneously and has good specificity and sensitivity. The method may provide potential for rapid surveillance and differential diagnosis of ALV or other immunosuppressive diseases in poultry.