Development Of Liquid Chip Kitfor Six Immunosuppressive Avian Diseases

In this study,the six immunosuppressive avian diseases were used as the research object to establish a rapid,sensitive,accurate,simple,practical and high-throughput detection method,which named general multiplex RT-PCR integrated with luminex?GMPLex in abbreviation?.The major studies include:The type of 10 strainsof immunosuppressive avian virus from technology center of Shenzhen Enter-exit Inspection and Quarantine Bureau were verified with hemagglutination?HA?,hemagglutinationingibition?HI?and real time RT-PCR method.The ELD₅₀value for A/C hicken/Hongkong/SZ-HI/1997?H5N1?and NDV-F48E9 were calculated through Reed-Muench method based on the spec ific death number of chicken embryos by HA and HI tests,and the results were 10^-7.75/0.2mL and 10^-8.5/0.2mL respectively.Specific universal super primers and probes were separately introduced to AIV-M,NDV-F,IBDV-VP2,MDV-PP38,ALV-J-ENV,CAV-VP2 to establish a multi-target universal amplification method at one time for six immunosuppressive avian virus,which named multiplex RT-PCR?GM RT-PCR?.The new GM RT-PCR amplification diagnosis technique which can fast identify diagnosis the positive strains of immunosuppressive avian virus,combined with the high-throughput diagnosis technology of liquid chip,established a rapid high-throughput diagnosis method named GMPLex for six immunosuppressive avian diseases.The GMPLex test presents the following features:high throughput,able to determine the types of 6 target genes for AIV,NDV,IBDV,MDV,ALV-J and CAV at one time;high specificity,ensured the specificity of the different types by using two nested degenerate primers and one probe,no cross reactio n occurring between the subtypes,no non-specific reactions with other pathogens and high sensitivity,When used separately to detect the strains of H5 and NDV,the GMPLex test showed a sensitivity of 10^-5(=280ELD₅₀)for both tests.When used to detect the 3 RN A strains,the GMPLex test showed a sensitivity of 10^-4(=2800ELD₅₀).The results of GMPLex comparison test of small samples shows that it's the same as the traditional methods.It was also proved that the GMPLex rapid high-throughput diagnosis test can satisfy the needs of high-throughput immunosuppressive avian virus at national ports.