Establishment And Preliminary Application Of Immuno-PCR Methods For Detecting H5N1 Avian Influenza Virus

Highly pathogenic avian influenza (HPAI) is an extremely contagious, multi-organ systemic disease of poultry leading to high mortality, and caused by some H5 and H7 subtypes of type A influenza virus. The accurate and prompt diagnosis of H5N1 infection in birds is a critical component of a disease control plan.To improve the ability to detect avian influenza virus (AIV) as well as for disease control, laboratories need more rapid and less cumbersome methods for the direct identification of the AIV H5 subtype in clinical samples.The immuno-polymerase chain reaction (immuno-PCR or IPCR) is a method that combines the specificity of immunologic detection methods with the exponential amplification of PCR. IPCR is a powerful tool and is one of the most sensitive detection methods for trace protein.In our study,we designed several IPCR methods for the ultra-low detection of H5 subtype AIV or H5 Hemagglutinin protein of AIV.From our experiments, the following results were obtained:(1) An indirect immuno-PCR assay and an indirect sandwich immuno-PCR assay, based on Immunology and polymerase chain reaction(IPCR) which combining enzyme immunoassay with DNA amplification ,was developed. Using the optimized concentration of reporter DNA and streptavidin, both indirect and indirect sandwich immuno-PCR assays detected AIV/H5 protein as low as 1 fg. These results are a nearly 1000-fold improvement over conventional indirect ELISA and indirect sandwich ELISA methods. Our data demonstrate that This MAb-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.(2) An immuno-PCR method for detection of H5 Hemagglutinin subtype avian influenza virus using magnetic gold particles as carriers was designed and carried out .This IPCR was able to detect as little as 10-4 EID50/mL H5 subtype AIV using a optimal concentration of reporter DNA and streptavidin. Our data demonstrated that this monoclonal antibody-based immuno-PCR method provides a platform capable of rapid screening of clinical samples for trace levels of AIV H5(3) Detecting avian influenza virus (AIV) and Newcastle disease virus (NDV) at low concentrations from tracheal and cloacal swabs of avian influenza- and Newcastle disease-infected poultry was carried out using a highly sensitive immunological-polymerase chain reaction (immuno-PCR) method. Magnetic gold particles were pre-coated with a capture antibody, either a monoclonal anti-AIV/H5 or monoclonal anti-NDV/F and viruses serially diluted ten-fold from 102–10-5 EID50 /mL. A biotinylated detection antibody bound to the viral antigen was then linked via a streptavidin bridge to biotinylated reporter DNA. After extensive washing, reporter DNA was released by denaturation, transferred to PCR tubes, amplified, electrophoresed and visualized. An optimized immuno-PCR method was able to detect as little as 10-4 EID50 mL-1 AIV or NDV.Our data demonstrated that this monoclonal antibody-based immuno-PCR method provides a platform capable of rapid screening of clinical samples for trace levels of AIV H5 and NDV in one step.(4) We designed a modified real-time IPCR method for the ultra-low detection of H5 subtype AIV. the TopYield microtiter plates were coated with ten- fold serial dilutions of AIV,which was recognized by mouse anti-AIV H5 monoclonal antibody that was directly linked with reporter DNA using succinimidyl 4- ( N-maleimidomethyl ) - cyclohexane-1-carboxylate (SMCC),a heterobifunctional cross-linker.The reporter DNA including only a BamH I- restriction site was released by a specific enzymatic restriction , then transferred to PCR tubes, amplified, and used as the signal for detection of AIV. Under the optimized condition ,MAb-based immuno- PCR(IPCR) method could measure 100μLof AIV H5N1 with 10-4EID50/ mL. The results showed that this modified IPCR was a highly specific assay for AIV H5 and had an approximately 1000-fold improvement over the conventional ELISA ,and a 100-fold enhancement compared with RT-PCR in detection sensitivity.In conclusion, by using TopYield plates or magnetic gold particles as the adsorption surface and using chemical material (SMCC) directly link reporter DNA to antibody ,or biotinylated detection antibody bound to the viral antigen linked via a streptavidin bridge to biotinylated reporter DNA ,We have developed four MAb-based IPCR for the detection of H5 subtype AIV with a detection limit as low as 10-4 EID50 /mL and I fg of H5 Hemagglutinin of AIV. These IPCR detection methods can be completed within 6 h, thus providing an alternative for the clinical detection of AIV H5 infections and offering a platform capable of mass screening of clinical samples .Therefore,These modified MAb-based immuno-PCR method are potential tools for the detection of ultra-low amount of pathogenic organisms and could serve as a model for other immuno-PCR assays.