Development of a Denaturing High-Performance Liquid Chromatography Method for Detection of Protist Parasites of Metazoans

Abstract

Increasingly, diseases of marine organisms are recognized as significant biotic factors affecting ecosystem health. However, the responsible disease agents are often unknown and the discovery and description of novel parasites most often rely on morphological descriptions made by highly trained specialists. Here, we describe a new approach for parasite discovery, utilizing denaturing high-performance liquid chromatography (DHPLC) reverse-phase ion-paring technology. Systematic investigations of major DHPLC variables, including temperature, gradient conditions, and target amplicon characteristics were conducted to develop a mechanistic understanding of DNA fragment separation by DHPLC. As a model system, 18S rRNA genes from the blue crab (Callinectes sapidus) and a parasitic dinoflagellate Hematodinium sp. were used. Binding of 18S rRNA gene PCR amplicons to the DNA separation column in the presence of triethylammonium acetate (TEAA) was inversely correlated with temperature and could be predicted based on the estimated DNA helicity of the PCR amplicon. Amplicons of up to 498 bp were resolved as single chromatographic peaks if they had high (>95%) DNA helicity. Amplicons that differed by as few as 2 bp could be resolved. Separation of 18S rRNA gene PCR amplicons was optimized by simultaneous manipulation of both temperature and solvent gradients. The optimal conditions included targeting regions of high DNA helicity (>95%), temperatures in the range of 57 to 63°C, and a linear acetonitrile gradient from 13.75 to 17.5% acetonitrile in 0.1 M TEAA (55 to 70% buffer B) over a 9-min period. Under these conditions, amplicons from a variety of parasites and their hosts can be separated and detected by DHPLC.

The influence of vent systems on pelagic eukaryotic micro-organism composition in the Nordic Seas

“…Extraction was carried out according to the manufacturer's instructions. PCR was conducted using the Takara Ex HS taq (Takara BIO, Inc., Japan) with the universal eukaryotic 18S rRNA gene-targeted primers Univ-1131F-7 (5 0 -AAA CTT AAA GRA ATT GAC GG-3 0 ) and Univ-1428R (5 0 -CTA AGG GCA TCA CAG ACC-3 0 ) bp (Troedsson et al 2008;Hadziavdic et al 2014) generating a *315-bp product. The amplification conditions included an initial denaturation cycle (3 min at 94°C), followed by 30 amplification cycles (30 s at 94°C, 30 s at 57.4°C and 1 min at 72°C).…”

Development of Clamping Probe for Rare DNA Detection using Universal Primers

“…For CS model, separation was optimized at 59.5˚C of oven temperature and 60-70% of linear gradient buffer B at 3.5 mL/min of flow rate (Troedsson et al, 2008a) while, for PP model, 57.5˚C and 62-68% at 0.45 mL/min (Cho, 2012). Retention time of each host and parasite peaks were highly reproducible (Fig.…”

“…For detection of rare gene, PCR amplicon was further separated by DNASep HT Cartridge (Transgenomic, Omaha, NE) with linear gradient elution of WAVE ® Optimized Buffers. Separation condition was tested by varying oven temperature and buffer B% (Troedsson et al, 2008a;Cho et al, Unpublished) and for 10 min. Chromatographic analysis was carried out by HSX-3500 fluorescence detector (Ex = 495 nm and Em = 537 nm) after SYBR Gold staining (Molecular Probe Inc.) with Navigator software version 1.6.2 (build 12) (Transgenomic, Omaha, NE).…”

“…To investigate the variation of clamping efficiency depending on clamping probe concentration, clamping-PCR was performed, with varying concentration (0 -2 μM) of each probes, by a set of universal primer: Univ18s-1131F (5´-AAA CTY AAAGRA ATT GAC CC-3´) (Troedsson et al, 2008a) and Univ18s-1629R (5´-GAC GGG CGG TGT GTR C-3´) (Gruebl et al, 2002). The twenty μl of PCR reaction mix was constituted by 1X Discoverase TM PCR buffer (Invitrogen), 0.3U of Discoverase TM DNA Polymerase (Invitrogen), 0.5 μM of each Primer, varying concentration of clamping probe, 200 μM of dNTP, 2 mM of MgSO 4 and 2 μl of templates.…”

“…Several targets are available for each taxonomy now using 16S rDNA for bacteria (Baker et al, 2003;Tian et al, 2005), 18S rDNA for eukaryote (Blankenship and Yayanos, 2005), 23S rDNA for marine algae and cyanobacteria (Sherwood and Presting, 2007), and 28S rDNA and ITS for fungi (Mohlenhoff et al, 2001;Iwen et al, 2002). In spite of convenience of the universal primer with ease of application to most species, separation of the amplicons from unknown sample mostly relied on a special treatment such as restriction enzyme (Lin et al, 2004) and denaturing high performance liquid chromatograph (DHPLC) (Troedsson et al, 2008a;Troedsson et al, 2008b). Universal primer, however, have a drawback such as underestimation of rare parasites in parasitic diagnosis because of a biased amplification of dominant gene (Bass and Cavalier-Smith, 2004).…”