Application of a real-time PCR assay to detect and quantify the myxozoan parasite Ceratomyxa shasta in river water samples
Application of a real-time PCR assay to detect and quantify the myxozoan parasite Ceratomyxa shasta in river water samples
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DOI：
10.3354/dao071109
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发表时间：
2006-07-25
影响因子：
1.4
通讯作者：
Bartholomew, Jerri L.
中科院分区：
文献类型：
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作者：
Hallett, Sascha L.;Bartholomew, Jerri L.
沙斯塔角潜蝇（Ceratomyxashasta）是一种鲑科鱼类的致病性病原菌，广泛分布于北美西北太平洋地区。目前的寄生虫检测方法涉及哨兵鱼暴露，既费力又耗时。作为替代，过滤协议和定量实时TaqMan聚合酶链反应（QPCR）检测和计数寄生虫孢子在河水中开发。荧光检测到寄生虫的粘孢子和放线孢子阶段，但不是从鱼或多毛类宿主或从9个其他粘虫测试。检测到小于1/1000的孢子，表明每个孢子具有> 1000个拷贝的靶18 S rRNA基因。该测定法在11份河水中检测到1份孢子。通过减少模板体积并在反应中加入牛血清白蛋白，克服了一些河流样品对测定的抑制作用;偶尔需要第二步纯化。利用QPCR方法研究了俄勒冈州/加州的克拉马斯河中沙斯塔虫的时空分布。在整个河流中检测到寄生虫，5条支流中有2条对干流有寄生虫。QPCR循环阈值与已知的完整孢子起始数的标准曲线的相关性揭示了寄生虫丰度超过20个孢子l（-1）的几个位点。虽然四次PCR数据证实了哨鱼接触的结果，但孢子数量与接触组的死亡率并不一致。水采样和过滤协议结合的QPCR检测是一种简单，相对快速的方法检测和定量的寄生虫水平的环境水样。
Ceratomyxa shasta is a virulent pathogen of salmonid fishes that is enzootic in the Pacific Northwest of North America. Current parasite detection methods involve sentinel fish exposures that are laborious and time-consuming. As a substitute, a filtering protocol and a quantitative real-time TaqMan polymerase chain reaction (QPCR) assay were developed to detect and enumerate parasite spores in river water. Fluorescence was detected from both the myxospore and actinospore stages of the parasite but not from the fish or polychaete hosts or from 9 other myxozoans tested. Less than 1/1000th of a spore was detected, indicating each had > 1000 copies of the target 18S rRNA gene. The assay detected 1 spore in 1 1 river water. Inhibition of the assay by some river samples was overcome by reducing the template volume and including bovine serum albumin in the reaction; occasionally a second purification step was required. The QPCR methodology was utilised to investigate the temporal and spatial distribution of C, shasta in the Klamath River, Oregon/California. The parasite was detected throughout the river, and 2 of 5 tributaries tested contributed parasites to the mainstem. Correlation of QPCR cycle threshold values with a standard curve for known starting numbers of whole spores revealed several sites where parasite abundance was in excess of 20 spores l(-1). Although QPCR data corroborated results of sentinel fish exposures, spore numbers did not correlate consistently with mortality in the exposure groups. The water sampling and filtering protocol combined with the QPCR assay is a simple and relatively rapid method for detection and quantification of parasite levels in environmental water samples.