The Study On The Detection Assays Of Sandwich ELISA, Colloidal Gold Strip And PCR For EDS-76 Virus

Egg drop syndrome-76 virus (EDS-76 virus) was extracted with chloroform and concentrated with polyethylene glycol-6000 (PEG-6000). Hyper immune sera of chicken anti-EDS-76 virus and rabbit anti-EDS-76 virus were prepared by immunizing in chicken and rabbit with purified EDS-76, Sheep anti-rabbit serum was obtained by immunizing in sheep with healthy rabbit immunoglobulin G (IgG). The purify serum were obtained by caprilic acid-ammonium sulfamate precipitation.A Antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed for the detection of EDS-76 virus by employing the three kinds of IgG The optimal dilutions of chicken anti-EDS-76 virus, rabbit anti-EDS-76 virus and HRP-sheep anti-rabbit were 1:160, 1:300 and 1:1000, respectively. The result showed that it could detect minimal to 5 ng/well of the purified EDS-76 virus antigen. The detection to the sample from experimental infected chickens and the clinical cases showed that the method of antibody sandwich ELISA has high sensitivity, good specify, and is suitable to test large numbers of samples.The colloidal gold particles were synthesized by reducing hydrogen tetrechloroaurate with sodium citrate. A colloidal gold particle of 15 nm in diameter was selected as labeling according to the different colors showed in detection. The minimum of labeling antibody was 40μg/mL. The optimal amount of chicken anti-EDS-76 virus and sheep anti-rabbit IgG was determined as 1μg. Meanwhile, the colloidal gold strip could detect as little as 1.35μg/mL of the purified EDS-76 virus.Polymerize chain reaction (PCR) assay was employed for detection of EDS-76 virus. A pair of primers was synthesized based on the DNA sequence of EDS-76 virus. An expected of 300bp DNA fragment of EDS-76 virus was amplified by PCR using the primers. The PCR assay could detect as little as 2.5×10 2pg of purified DNA of EDS-76 virus. The crossing-reaction and sensitive-reaction indicated that PCR is sensitive and specific. The PCR test showed that the EDS-76 does not exist in the heart, exist during 7-15 day post infection (DPI) in liver and kidney, 4-21 DPI in uterus and blood and 4-17 DPI in spleen. It is an effective method used to diagnose EDS-76 virus.The 135 samples collected from uteri in natural infectious chickens were detected...