Rapid Detecting High Pathogenic Avian Influenza (HPAIV) Virus Subtype H5 By Real Time RT-PCR

For further improving the sensitivity and specificity of detection of HPAIV subtype H5 , the real-time RT- PCR (RRT-PCR) was developed in this study using the high sensitivity and obturation of RRT-PCR to shortening the detection time and lowering the storage cost of poultry products. Based on the conservative sequence of haemaggIutinin (HA) of subtype H5 of HPAIV, 70 pairs of primer and 4 probes were designed and synthesized, a RRT-PCR was established for direct detecting the high pathogenic avian influenza virus subtype H5 (HPAIV H5) from poultry meat by series of experiments including the selection and optimization of primers and probes, reverse transcriptase , Taq Polymerase and concentration of ions etc.. The sensitivity of the method for detecting HPAIV H5 was determined as TO-6-10-7approximately by testing the infective allantoic fluid of four HPAIV H5 strains with diluted serially , The high specificity of the method was demonstrated by detecting Chicken anaemia virus, Infectious bursal disease virus, Infectious bronchitis virus (H120, H52 and nephropathogenic strains) , velogenic strains and vaccines of Newcastle disease virus, subtype H9 of Avian influenza virus, Duck plague virus and Duck viral hepatitis virus, which could be potentially infected in chicken and duck products during feeding. Compared with the international reference standard method - virus isolation(VI) with embryonated eggs designated by OIE, the detection time was shortened consumedly from originally at least 21 days for embryo isolation to 4 hours.The 20 SPF chickens, 30 broilers and 30 ducks were artificially infected with four HPAIV H5 strains respectively. All broilers died in 7 PDI. The death rate of SPF chickens was 80-90%(8-9/10) in 5 DPI. The death rate of duck was 6.7% (2/30) , and most of infected ducks were not shown any clinical signs in 23 DPI. Comparative detection of RRT-PCR and embryo isolation(EI) was carried out through the tissues homogenates diluted serially of heart, liver, spleen, lungs, kidneys, trachea, rectum, marrow, muscle of chest, legs and the wings, which sampled from the dead broilers, SPF chickens and ducks. The detection limit of RRT-PCR and EI was 10-4and 10-4~10-5 for muscle tissues of SPF chickens, but 10-3~l0-5 and 10-3-10-5 for internal organs from SPF chickens respectively. The detection limit of RRT-PCR and EI was 10-3~10-4 and 10-4~10-5 for muscle tissues of broilers, but 10-2~10-5 and 10-3~10-6 for internal organs of broilers respectively. The detection limit of RRT-PCR and EI was 10-2~10-4 and 10-2~10-3 for muscle tissues of died ducks, but original suspension~10-5 and negative original suspension ~10-4 for internal organs of died ducks respectively. The detective sensitivity of two methods was unobvious various by statistical analysis. The ducks artificially infected with no any clinic signs were slaughtered at different time. The samples of muscle and viscera were tested by both RRT-PCR and VI for the presence of HPAIV H5. The result suggested all viscera samples were positive at 3 DPI, the virus of viscera in some ducks were decreased gradually and some were negative at 9-15 DPI by two methods. But the virus of viscera was increased at 23 DPI.Comparative detection of RRT-PCR and EI for the muscle tissues and internal organs from natural infected ducks showed the results inconsistent for different individuals. Butfor same sample, the difference between two methods is unobvious (P >0.05) .