Development of a Denaturing High-Performance Liquid Chromatography (DHPLC) Assay to Detect Parasite Infection in Grass Shrimp Palaemonetes pugio

Abstract

In developing a useful tool to detect parasitic dynamics in an estuarine ecosystem, a denaturing high-performance liquid chromatography (DHPLC) assay was optimized by cloning plasmid DNA from the grass shrimp Palaemonetes pugio, and its two parasites, the trematode Microphallus turgidus and bopyrid isopod Probopyrus pandalicola. The optimal separation condition was an oven temperature of 57.9°C and 62-68% of buffer B gradient at a flow rate of 0.45 mL/min. A peptide nucleic acid blocking probe was designed to clamp the amplification of the host gene, which increased the amplification efficiency of genes with low copy numbers. Using the DHPLC assay with wild-type genomic, the assay could detect GC Gram positive bacteria and the bopyrid isopod (P. pandalicola). Therefore, the DHPLC assay is an effective tool for surveying parasitic dynamics in an estuarine ecosystem.

Molecular features ofProbopyrussp. (Isopoda: Bopyridae) from Brazilian Amazonia and the parasitism of inland populations ofMacrobrachium amazonicum(Decapoda: Palaemonidae)

“…(2013) expanded the dataset to the superfamilies Bopyroidea and Cryptoniscoidea. In the specific case of the genus Probopyrus , 18S rDNA sequences are available for P. pacifiensis (Dreyer and Wägele, 2001), P. buitendijki (Boyko et al ., 2013) and P. pandalicola (Cho, 2012), in addition to those described in the present study. Wu et al .…”

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.…”