Molecular Epidemiology Survery Of RA And Researches Of The Dynamic Distribution Regular Pattern In Ducks Artificially Infected With RA

In this study, a specific PCR assay was established using the 16Sr DNA sequences of RA. It can amplify a anticipative fragment about 838bp from RA, but we cann't get the same fragment from Escherichia Genus, Salmonella Genus, Staphylococcus Genus, Lactobacillus, Bifidobacteriμm, Bacillus and Enterococcμs. The sensitivity of the assay to detect least amount was 20 fg RA-DNA.Using the PCR method developed above, a molecular epidemiology survery of RA in ducks and conditions was done in Sichuan province. The results indicated that the important place with RA was the health duck, feed, drinking water, underlay grass and so on.A rapid Fluorescence quantitative PCR(FQ-PCR) assay was established using the capsular polysaccharide protein gene of RA(DQ151838) discovered in our laboratory. The assay contains a pare of primers and an internal dual-labled Fluorescence probe. The expression of the assay was Y=-3.416X +39.492, the correlation coefficient was 1.000 and PCR efficiency reached to 96.2%. there was excellent linear during the DNA concentration from 2.0 to 2.0×10~8 copies. The sensitivity of the assay to detect least amount was 2.0 copies RA-DNA(equal to 1.0 CFU RA).it was 10~4 times higher than the PCR assay established in my thesis. A negative result appeared for detecting Escherichia Genus, Salmonella Genus and Staphylococcus Genus. The positive coincidence rate when compare RA isolation with FQ-PCR to detect the samples of heart, liver and brain from died ducks experimental infected with RA was 100 percent. When to detect the samples of heart, liver and brain from died ducks with RA typical clinical symptom and pathologic change from field, the positive coincidence rate of FQ-PCR was observably higher than that of isolation.Seven-day-old ducks infected with 1/10 LD50 RA, and the results indicated that when two hours after inoculation, RA-DNA were detected in heart, liver, lung, thymus, esophagus by Subcutaneous infection, in heart, liver, lung, thymus, esophagus, trachea, duodenum, pharyngeal swab by nasally infection, and in esophagus, trachea, duodenum, pharyngeal swab by orally infection. four hours after inoculation, in addition to brain and blood (RA-DNA wasn't also detected in pancreas by orally infection), RA-DNA were detected in all samples. Eight hours after inoculation, all the samples were positive in addition to brain by orally infection. RA-DNA in heart, liver, thymus were the highest in all samples, next was lung, kidney, spleen, bursa of fabricius. Brain and pancreas had a low quantity of RA in ducks.