Preferential PCR amplification of parasitic protistan small subunit rDNA from metazoan tissues

The Journal of Eukaryotic Microbiology
Susan M BowerMichelle W Mak

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...Continue Reading

References

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Jun 14, 2003·Diseases of Aquatic Organisms·Ryan B CarnegieSusan M Bower

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Citations

Oct 5, 2015·Trends in Parasitology·David BassHanna Hartikainen
Aug 15, 2014·Environmental Microbiology·Marianne SimonPurificación López-García
Jan 16, 2013·The Journal of Eukaryotic Microbiology·Fumiya NoguchiKiyotaka Takishita
Jun 16, 2016·Frontiers in Microbiology·Marianne SimonLudwig Jardillier
May 11, 2017·Environmental Microbiology·Aurélien SaghaïPurificación López-García
Jul 7, 2019·Environmental Microbiology·Javier Del CampoPatrick J Keeling
Apr 5, 2019·Nature·Waldan K KwongPatrick J Keeling
Sep 29, 2020·Environmental Microbiology·Miguel IniestoPurificación López-García
Feb 28, 2021·Molecular Ecology·Gwendoline M DavidLudwig Jardillier
Aug 1, 2020·European Journal of Protistology·David Bass, Javier Del Campo
Mar 24, 2021·Molecular Ecology·Albert ReñéEsther Garcés
Sep 23, 2021·Molecular Ecology Resources·Diana MinardiDavid Bass

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Methods Mentioned

BETA
PCR
electrophoresis
electron microscopy

Software Mentioned

UNonMet
BLAST

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