Abstract:

The primers and probe were designed according to Riemerella anatipestifer (RA) outer membrane protein (ompA) gene conserved sequence from the GenBank and a real-time fluorescence qPCR quick detection method of RA was established. The detection limit of the method was 8.0 copies/μL of RA DNA. It had a high specificity, was negative to the duck-origin E. coli, Pasteurella multocida, Salmonella, Proteus, Staphylococcus aureus. Ducklings artifically infected with a RA strain, and throat swabs, cloacal swabs, blood, liver, lung and brain were collected ,detected by real-time qPCR and isolated 〖HT〗〖ST〗〖WT〗〖HJ〗

〖WT5”BZ〗pathogenic bacteria. The results of 1/10 half lethal dose inoculation group showed that RA DNA in liver and brain were detected 8 h after infection, all the samples were positive 16 h after, RA were first isolated from liver 24 h after, RA positive rate was 63.8% (51/80) by real-time qPCR while the RA isolation rate was 28.8% (23/80) only. The results of 10 half lethal dose inoculation group showed that RA DNA in throat swab, heart and liver were detected 1 h after infection, all the samples were positive at 5 h, RA were first isolated from the lungs and brain at 5 h, RA positive rate was 86.3% (69/80) by real-time qPCR while the RA isolation rate is 60% (48/80) only. It was only 30 min for extracting RA DNA from detection tissue by pyrolysis. The detection time of the established real-time qPCR method was only 2 h.The method could be used for rapid diagnosis , epidemiological investigation, quarantine of introduction ducks, import and export inspection and quarantine of live ducks and their products.

Key words: Riemerella anatipestifer; real-time qPCR; rapid detection; sensitivity; specificity