Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia
Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia
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DOI：
10.3354/dao042199
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发表时间：
2000-09-28
影响因子：
1.4
通讯作者：
Distel, DL
中科院分区：
文献类型：
--
作者：
Carnegie, RB;Barber, BJ;Distel, DL
发展比传统组织学技术更敏感和特异的诊断方法对扁牡蛎博那线虫病的管理非常重要。开发了一种特异性聚合酶链反应（PCR）方案，用于检测牡蛎鳃和血淋巴中大量DNA中的极少量Bonamia ostrae（Pichot et al. 1980）核糖体DNA（rDNA）。760 bp的PCR扩增产物的存在对应于B，牡蛎感染细胞学确定在185牡蛎从爱尔兰，西班牙和美国。所有（100%）的“重度”和“中度”感染的牡蛎，86.7%的“轻度”感染的牡蛎，和66.7%的“几乎”感染的牡蛎被证实感染使用PCR。此外，37.9%的牡蛎中含有B。细胞学检测未检出的牡蛎，PCR检测均为阳性。采样误差和细胞学诊断的主观性是诊断方法之间的分歧的可能来源非常轻的感染牡蛎。与常规的组织学和细胞学技术相比，PCR技术具有更高的灵敏度和更少的模糊性。DNA序列数据的系统发育分析证实了B，ostrace是Haplosporidia的成员。
The development of diagnostic assays more sensitive and specific than traditional histological techniques is important for the management of bonamiasis in flat oysters Ostrea edulis. A specific polymerase chain reaction (PCR) protocol was developed for the detection of very small amounts of Bonamia ostreae (Pichot et al. 1980) ribosomal DNA (rDNA) in bulk DNA from oyster gill and hemolymph. The presence of a 760 bp PCR amplification product corresponded with B, ostreae infections determined cytologically in 185 oysters from Ireland, Spain, and the USA. All (100%) 'heavily' and 'moderately' infected oysters, 86.7% of the 'lightly' infected oysters, and 66.7% of the 'scarcely' infected oysters were confirmed to be infected using the PCR. In addition, 37.9% of the oysters in which B. ostreae was not detected using cytology were positive using the PCR. Sampling error and the subjectivity of cytological diagnoses are the likely sources of disagreement between diagnostic methods in oysters with very light infections. The PCR assay developed here is more sensitive and less ambiguous than standard histological and cytological techniques, Phylogenetic analysis of DNA sequence data confirmed B, ostreae to be a member of the Haplosporidia.