Polymerase chain reaction for the detection of Newcastle disease virus

Yeo, Sang-geon; Kim, Do-kyoung; Park, Seon-ja

Korean Journal of Veterinary Research 1998;38(3):565-573.

Sang-geon Yeo¹, Do-kyoung Kim², Seon-ja Park³

¹Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University
²Kyongnam Livestock Promotion Institute
³Department of Biology, Graduate School, Gyeonsang National University

닭 뉴캐슬병 바이러스의 특이 검출을 위한 polymerase chain reaction 법

여상건¹, 김도경², 박선자³

¹경상대학교 수의과대학 동물의학연구소
²경남축산진흥연구소
³경상대학교 대학원 생물학과

Abstract

To study the specific tools for the diagnosis of Newcastle disease virus (NDV) in chicken, polymerase chain reaction (PCR) and its presumable conditions were evaluated for the detection of hemagglutinin-neuraminidase (HN) gene of NDV RNA. For these purposes, Kyojeongwon strain of the NDV was propagated in allantoic cavity of SPF embryonating chicken eggs, and viral RNA was extracted from fractionated virus after the allantoic fluids were ultracentrifuged with sucrose gradient. The first-strand cDNA was then made for the HN gene of NDV RNA by reverse transcription at $42^{circ}C$ for 1 hour using specific primer complementary to the HN gene. The single-stranded cDNA was used as template in the PCR of the HN-DNA, and various conditions of the PCR were evaluated to set up method for the specific detection of the HN-DNA. The PCR conditions promising for the detection of HN gene consist of preheating at $94^{circ}C$, 5 min, 30 cycles of denaturation at $94^{circ}C$, 1 min, annealing at $55^{circ}C$, 1 min and polymerization at $72^{circ}C$, 2 min, and a cycle of extension at $72^{circ}C$, 5 min. when NDVs of allantoic fluids without fractionation were applied to the above PCR condition, the HN genes were detected effectively not only from Kyojeongwon but from other velogenic strains such as Herts and a field isolate.

Key Words: Newcastle disease virus, HN gene, reverse transcription, PCR