Preferential PCR Amplification of Parasitic Protistan Small Subunit rDNA from Metazoan Tissues

Abstract

A "universal non-metazoan" polymerase chain reaction (UNonMet-PCR) that selectively amplifies a segment of nonmetazoan Small Subunit (SSU) rDNA gene was validated. The primers used were: 18S-EUK581-F (5'-GTGCCAGCAGCCGCG-3') and 18S-EUK1134-R (5'-TTTAAGTTTCAGCCTTGCG-3') with specificity provided by the 19-base reverse primer. Its target site is highly conserved across the Archaea, Bacteria, and eukaryotes (including fungi), but not most Metazoa (except Porifera, Ctenophora, and Myxozoa) which have mismatches at bases 14 and 19 resulting in poor or failed amplification. During validation, UNonMet-PCR amplified SSU rDNA gene fragments from all assayed protists (n = 16 from 7 higher taxa, including two species of marine phytoplankton) and Fungi (n = 3) but amplified very poorly or not at all most assayed Metazoa (n = 13 from 8 higher taxa). When a nonmetazoan parasite was present in a metazoan host, the parasite DNA was preferentially amplified. For example, DNA from the parasite Trypanosoma danilewskyi was preferentially amplified in mixtures containing up to 1,000 x more goldfish Carassius auratus (host) DNA. Also, the weak amplification of uninfected host (Chionoecetes tanneri) SSU rDNA did not occur in the presence of a natural infection with a parasite (Hematodinium sp.). Only Hematodinium sp. SSU rDNA was amplified in samples from infected C. tanneri. This UNonMet-PCR is a powerful tool for amplifying SSU rDNA from non-metazoan pathogens or symbionts that have not been isolated from metazoan hosts.

Retracted: Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidianEnterocytozoon hepatopenaeiin penaeid shrimp

“…These problems can be solved by the development of rapid and sensitive molecular detection methods that allow for real-time detection of carrier states in cultivated shrimp and in potential reservoir species in the culture environment. These alternatives to histological detection of parasites are often based on detection of species-specific nucleic acid sequences of small subunit (SSU) rRNA genes Bower et al 2004;Ndao 2009). For this purpose, traditional PCR or RT-PCR detection methods are restrictive because they require high investment in equipment (e.g.…”

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

“…Although blocking probes can suppress the amplification of host gene fragments, this does not mean that the predominant host gene is not amplified. To overcome this problem, selective targeting of universal primers or nonphylum universal primers (Bower et al, 2004), which amplify only parasites or weakly including target species, can be considered. Several phylum-specific universal primers have been developed and extensively used not only for DHPLC assays but also for several gel electrophoresis-related methods (Iwen et al, 2002;Baker et al, 2003;Blankenship and Yayanos, 2005;Sherwood and Presting, 2007).…”

“…certain species with high sensitivity. To overcome these drawbacks, universal primers have been developed to target SSU rDNA of diverse phylum (Bower et al, 2004;Blankenship and Yayanos, 2005). When coupled with universal primers, DH-PLC can be a competent method due to its high sensitivity for separation, which can detect as small as a 2-bp difference in sequence composition (Troedsson et al, 2008b).…”

Haplosporidium littoralis sp. nov.: a crustacean pathogen within the Haplosporida (Cercozoa, Ascetosporea)

“…Renault et al 2000) were unsuccessful. PCR was then attempted with primers 18S-EUK581f (5'-GTG CCA GCA GCC GCG-3') and 18S-EUK 1134r (5'-TTT AAG TTT CAG CCT TGC G-3'), which were designed to selectively amplify approximately 550 bp from the middle section of SSU rDNA of nonmetazoans (Carnegie et al 2003, Bower et al 2004. Reactions contained buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.0 mM MgCl 2 , 0.001% gelatin; Applied Biosystems), each deoxyribonucleotide at 0.2 mM, 0.4 µg bovine serum albumin, each primer at 12.5 pmol, 0.6 U AmpliTaq DNA polymerase (Applied Biosystems), and 200 to 250 ng DNA.…”