Detection of Infectious Bursal Disease Virus RNA by Reverse Trans cription -Polymerase Chain Reaction

• Main Authors: hiau Yea Tsong, 蕭雅聰
• Other Authors: Lee Long Huw
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/00570968485054590509

碩士 === 國立中興大學 === 獸醫學系 === 82 === The reverse transcription-polymerase chain reaction ( RT-PCR ) was used to detect Infectious bursal disease virus ( IBDV ) RNA. In the part of IBDV genome,two sets of primes detect standard strain. The specific primers were decided to the presented nucleotide sequence analysis of genome segment A of IBDV with artificial synthesis. The cDNA was synthesized at $45^\circ C$ ,60 minutes. The first amplification was proceeded 30 cycles with $92^\circ C 1$ minute, $35^\circ C$ 1 minute, $72^\circ C 2$ minutes. The second amplification condition is the same as the first amplification condition. The RT-PCR product was observed on 2.5\% agarose contained ethidium bromide . RT-PCR product were identified by using restriction endonucleases.The two sets of primers were selected and used as the detection of IBDV RNA. The sensitivity test showed that the lower limit of detection was approximately $10^{1.5} TCID_{50 }$ of IBDV . The primers did not react with nucleic acids of chicken bursal cell, chicken spleen cell, chicken embryo fibroblast cell, fowlpox virus, Marek's disease virus, turkey herpes virus, avain reovirus, avain influenza virus, infectious bronchitis and infectious laryngotracheitis virus. In addition to yhe detection of IBDV RNA of bursas, buffycoat collected from the chickens experimentally exposed to IBDV, the primers were also used to detect the viral RNA extracted from spleens of spadger and chicken bursas in several poultry farms where the IBD infections were suspected.