Spackman E, Senne D A, Bulaga L L, Trock S, Suarez D L
Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 934 College Station Road, Athens, GA 30605, USA.
Avian Dis. 2003;47(3 Suppl):1087-90. doi: 10.1637/0005-2086-47.s3.1087.
A multiplex real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) assay for the simultaneous detection of the H5 and H7 avian influenza hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) reporter dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro transcribed RNA templates, to have a reproducible detection limit for H7 of approximately 10(4) HA gene copies and approximately 10(4)-10(5) HA gene copies of H5. A direct comparison of H5-H7 multiplex RRT-PCR with hemagglutination inhibition (HI) was performed with 83 AI RRT-PCR and virus isolation positive tracheal and cloacal swab samples obtained from various avian species and environmental swabs from live-bird markets in New York and New Jersey. Both multiplex RRT-PCR and HI agreed on the subtype determination of 79 (95.2%) of the 83 samples, of which 77 were positive for H7 and two were determined to be non-H5/non-H7 subtypes. No samples were determined to be the H5 subtype by either assay.
开发了一种多重实时逆转录-聚合酶链反应（RRT-PCR）检测方法，用于同时检测H5和H7禽流感血凝素（HA）亚型，该方法采用了分别标记有FAM（H5探针）和ROX（H7探针）报告染料的水解型探针。使用体外转录的RNA模板确定了H5-H7亚型分型检测方法的灵敏度，结果显示对H7的可重复检测限约为10⁴个HA基因拷贝，对H5的可重复检测限约为10⁴ - 10⁵个HA基因拷贝。使用从纽约和新泽西的各种禽类以及活禽市场环境拭子中获得的83份禽流感RRT-PCR和病毒分离阳性的气管和泄殖腔拭子样本，对H5-H7多重RRT-PCR与血凝抑制试验（HI）进行了直接比较。在83份样本中，多重RRT-PCR和HI对79份（95.2%）样本的亚型判定结果一致，其中77份为H7阳性，两份被判定为非H5/非H7亚型。两种检测方法均未将任何样本判定为H5亚型。
