Troedsson Christofer, Lee Richard F, Walters Tina, Stokes Vivica, Brinkley Karrie, Naegele Verena, Frischer Marc E
Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, GA 31411, USA.
Appl Environ Microbiol. 2008 Jul;74(14):4346-53. doi: 10.1128/AEM.02132-07. Epub 2008 May 23.
Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence information on eukaryotic parasites is not required. This distinction provides a significant advantage over specifically targeted PCR assays that do not allow for the detection of unknown or unsuspected parasites. However, initial field evaluations of the DHPLC assay suggested that because of PCR-biased amplification of dominant host genes it was not possible to detect relatively rare parasite genes in infected crab tissue. Here, we describe the use of a peptide nucleic acid (PNA) PCR hybridization blocking probe in association with DHPLC (PNA-PCR DHPLC) to overcome inherent PCR bias associated with amplification of rare target genes by use of generic primers. This approach was utilized to detect infection of blue crabs (Callinectes sapidus) by the parasitic dinoflagellate Hematodinium sp. Evaluation of 76 crabs caught in Wassaw Sound, GA, indicated a 97% correspondence between detection of the parasite by use of a specific PCR diagnostic assay and that by use of PNA-PCR DHPLC. During these studies, we discovered one crab with an association with a previously undescribed protist symbiont. Phylogenetic analysis of the amplified symbiont 18S rRNA gene indicated that it is most closely related to the free-living kinetoplastid parasite Procryptobia sorokini. To our knowledge, this is the first report of this parasite group in a decapod crab and of this organism exhibiting a presumably parasitic life history.
最近，我们描述了一种新型变性高效液相色谱（DHPLC）方法，该方法可用于初步检测和鉴定甲壳类寄生虫。由于此方法使用靶向18S rRNA基因保守区域的通用引物，因此不需要真核寄生虫的先验遗传序列信息。这一区别相对于特异性靶向PCR检测具有显著优势，后者无法检测未知或未怀疑的寄生虫。然而，DHPLC检测的初步现场评估表明，由于宿主优势基因的PCR偏向性扩增，无法在受感染的蟹组织中检测到相对罕见的寄生虫基因。在此，我们描述了一种肽核酸（PNA）PCR杂交阻断探针与DHPLC（PNA-PCR DHPLC）联合使用的方法，以克服使用通用引物扩增罕见靶基因时固有的PCR偏向性。该方法用于检测寄生性甲藻Hematodinium sp.对蓝蟹（Callinectes sapidus）的感染。对在佐治亚州瓦索湾捕获的76只螃蟹的评估表明，使用特异性PCR诊断检测法和使用PNA-PCR DHPLC检测寄生虫的结果之间的对应率为97%。在这些研究中，我们发现了一只与先前未描述的原生生物共生体有关联的螃蟹。对扩增的共生体18S rRNA基因的系统发育分析表明，它与自由生活的动基体寄生虫Procryptobia sorokini关系最为密切。据我们所知，这是该寄生虫类群在十足目螃蟹中的首次报道，也是该生物呈现出可能寄生生活史的首次报道。
