title: Development of one-step reverse transcriptase-polymerase chain reaction to detect duck hepatitis virus type 1.
abstract: A one-step reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed and optimized for the detection of duck hepatitis virus type 1 (DHV-1) using the Viral Gene-spin viral DNA/RNA extraction kit. A pair of DHV-1-specific primers was designed against the gene encoding RNA-dependent RNA polymerase (3D gene). Using RNA prepared from duckling liver samples infected with two reference and seven Korean field isolates of DHV-1, one-step RT-PCR with DHV1-specific primers amplified a 467-bp fragment. Under the same conditions, no amplification was observed for 14 other avian pathogenic viruses and bacteria. Using RNA prepared from serial dilutions of the DHV-1 with the supernatant of the uninfected duckling liver homogenate (10% w/v), the one-step RT-PCR assay was found to be sensitive to 10 50% egg lethal dose (ELD50) 0.1 ml(-1) of DHV-1. Furthermore, this method detected DHV-1 from the livers and allantoic fluid of duck embryos dying before 3 days postinoculation (PI) and of chicken embryos that were chilled at 3 days PI. Therefore, this one-step RT-PCR method is rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.
authors.affiliation: National Veterinary Research and Quarantine Service, 480 Anyang 6 dong, Manan-gu, Anyang, Gyeonggi-do, 420-824, Republic of Korea.
authors.forename: Min-Chul
authors.initials: MC
authors.lastname: Kim
authors.forename: Yong-Kuk
authors.initials: YK
authors.lastname: Kwon
authors.forename: Seong-Joon
authors.initials: SJ
authors.lastname: Joh
authors.forename: Jun-Hun
authors.initials: JH
authors.lastname: Kwon
authors.forename: Jae-Hong
authors.initials: JH
authors.lastname: Kim
authors.forename: Sun-Joong
authors.initials: SJ
authors.lastname: Kim
mesh_terms: D000818:Animals; D001483:Base Sequence; D004372:Ducks / Q000196:embryology / Q000821:virology; D004371:Hepatitis Virus, Duck / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity