Li Caiwen, Shields Jeffrey D, Small Hamish J, Reece Kimberly S, Hartwig Carmony L, Cooper Roland A, Ratzlaff Robert E
Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, Virginia 23062, USA.
Dis Aquat Organ. 2006 Oct 27;72(3):185-92. doi: 10.3354/dao072185.
Panulirus argus Virus 1 (PaV1) is the first virus known to be pathogenic to a wild lobster. It infects the Caribbean spiny lobster P. argus from the Florida Keys, and has a predilection for juveniles. The monitoring of the virus in wild populations and study of its behavior in the laboratory require the development of reliable diagnostic tools. A sensitive and specific fluorescence in situ hybridization (FISH) assay was developed for detection of PaV1. The lower detection limit using a 110 bp DNA probe in a dot-blot hygridization for PaV1 DNA was 10 pg of cloned template PaV1 DNA and 10 ng of genomic DNA extracted from the hemolymph of diseased spiny lobster. The fluorescein (FITC)-labeled probe specifically hybridized to PaVl-infected cells in the hepatopancreas, hindgut, gills, heart, foregut, and nerve tissues. FITC staining was observed around the inner periphery of the nuclear membrane, with lighter staining in a more dispersed pattern within the nucleus. The probe did not hybridize with host tissues of uninfected spiny lobsters, nor did it cross-react with 4 other virus samples tested. This assay will facilitate our understanding of the pathogenesis of the viral disease and help in monitoring efforts directed at determining the prevalence of PaV1 in juvenile nurseries for this lobster.
黄斑龙虾病毒1（PaV1）是已知的第一种对野生龙虾具有致病性的病毒。它感染来自佛罗里达群岛的加勒比刺龙虾（P. argus），并且偏好感染幼体。对野生种群中的该病毒进行监测以及在实验室中研究其行为需要开发可靠的诊断工具。为此开发了一种灵敏且特异的荧光原位杂交（FISH）检测方法用于检测PaV1。在斑点杂交中使用一条110 bp的DNA探针检测PaV1 DNA时，其最低检测限为10 pg克隆模板PaV1 DNA以及10 ng从患病刺龙虾血淋巴中提取的基因组DNA。异硫氰酸荧光素（FITC）标记的探针能与肝胰腺、后肠、鳃、心脏、前肠和神经组织中受PaV1感染的细胞特异性杂交。在核膜内周边观察到FITC染色，在细胞核内染色较浅且呈更分散的模式。该探针未与未感染刺龙虾的宿主组织杂交，也未与其他4种测试病毒样本发生交叉反应。该检测方法将有助于我们了解这种病毒性疾病的发病机制，并有助于监测旨在确定该龙虾幼体培育场中PaV1流行情况的工作。
