Development of one-step reverse transcriptase-polymerase chain reaction to detect duck hepatitis virus type 1.
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.
Kim
Min-Chul
MC
National Veterinary Research and Quarantine Service, 480 Anyang 6 dong, Manan-gu, Anyang, Gyeonggi-do, 420-824, Republic of Korea.
Kwon
Yong-Kuk
YK
Joh
Seong-Joon
SJ
Kwon
Jun-Hun
JH
Kim
Jae-Hong
JH
Kim
Sun-Joong
SJ
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