뉴캣슬병 바이러스 검출 및 병원성 감별을 위한 Duplex RT-PCR법 개발 (Development of a Duplex RT-PCR Assay for the Simultaneous Detection and Discrimination of Avirulent and Virulent Newcastle Disease Virus (NDV))

서지정보

· 발행기관 : 한국가금학회
· 수록지 정보 : 한국가금학회지 / 44권 / 2호 / 93 ~ 102페이지
· 저자명 : 김지예, 이현정, 장일, 이희수, 윤성준, 박지성, 설재구, 김승환, 홍지무, Zillian Wang, Hualei Liu, 최강석

초록

A duplex RT-PCR (dRT-PCR) assay was developed for the simultaneous detection and discrimination of nonvirulent and virulent Newcastle disease virus (NDV) in a single PCR tube. Primers targeting the large polymerase protein (L) gene and the fusion protein (F) gene of NDV were designed to detect all NDVs (by common type PCR primers) and virulent NDVs (by pathotype PCR primers), respectively and evaluated experimentally with reference NDV strains and other poultry viral pathogens. PCR products of the expected size of 386 bp were amplified from all NDV samples whereas PCR products of the expected size of 229 bp were amplified from virulent NDV samples alone. Cross reaction was not observed with other avian viral pathogens. The detection limit of NDV by the dRT-PCR was estimated to be 103 50% egg infectious dose/0.1 mL. In the dRT-PCR using field isolates of NDV, the pathotype PCR primers detected specifically all of virulent field isolates of NDV from Malaysia, Pakistan and China whereas common type PCR primers detected 94.4% (51/54) of field isolates of NDV from China. Three Chinese NDV isolates with false negative result were non-virulent viruses. Our results indicate that the dRT-PCR might provide a rapid and simple tool for rapid simultaneous detection and discrimination of non-virulent and virulent NDVs. Therefore the developed dRT-PCR assay provides a powerful novel means for the rapid diagnosis of Newcastle disease.

영어초록

A duplex RT-PCR (dRT-PCR) assay was developed for the simultaneous detection and discrimination of nonvirulent and virulent Newcastle disease virus (NDV) in a single PCR tube. Primers targeting the large polymerase protein (L) gene and the fusion protein (F) gene of NDV were designed to detect all NDVs (by common type PCR primers) and virulent NDVs (by pathotype PCR primers), respectively and evaluated experimentally with reference NDV strains and other poultry viral pathogens. PCR products of the expected size of 386 bp were amplified from all NDV samples whereas PCR products of the expected size of 229 bp were amplified from virulent NDV samples alone. Cross reaction was not observed with other avian viral pathogens. The detection limit of NDV by the dRT-PCR was estimated to be 103 50% egg infectious dose/0.1 mL. In the dRT-PCR using field isolates of NDV, the pathotype PCR primers detected specifically all of virulent field isolates of NDV from Malaysia, Pakistan and China whereas common type PCR primers detected 94.4% (51/54) of field isolates of NDV from China. Three Chinese NDV isolates with false negative result were non-virulent viruses. Our results indicate that the dRT-PCR might provide a rapid and simple tool for rapid simultaneous detection and discrimination of non-virulent and virulent NDVs. Therefore the developed dRT-PCR assay provides a powerful novel means for the rapid diagnosis of Newcastle disease.

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