Bower Susan M, Carnegie Ryan B, Goh Benjamin, Jones Simon R, Lowe Geoffrey J, Mak Michelle W
Fisheries and Oceans Canada, Pacific Biological Station, 3190 Hammond Bay Rd., Nanaimo, British Columbia V9T 6N7, Canada.
J Eukaryot Microbiol. 2004 May-Jun;51(3):325-32. doi: 10.1111/j.1550-7408.2004.tb00574.x.
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.
一种选择性扩增非后生动物小亚基（SSU）rDNA基因片段的“通用非后生动物”聚合酶链反应（UNonMet-PCR）得到了验证。所使用的引物为：18S-EUK581-F（5'-GTGCCAGCAGCCGCG-3'）和18S-EUK1134-R（5'-TTTAAGTTTCAGCCTTGCG-3'），其特异性由19个碱基的反向引物提供。其靶位点在古菌、细菌和真核生物（包括真菌）中高度保守，但在大多数后生动物（除海绵动物、栉水母动物和粘孢子虫外）中并非如此，这些后生动物在第14和19位碱基处存在错配，导致扩增不佳或失败。在验证过程中，UNonMet-PCR从所有检测的原生生物（n = 16，来自7个高级分类群，包括两种海洋浮游植物）和真菌（n = 3）中扩增出了SSU rDNA基因片段，但在大多数检测的后生动物（n = 13，来自8个高级分类群）中扩增非常差或根本没有扩增。当后生动物宿主中存在非后生动物寄生虫时，寄生虫DNA会被优先扩增。例如，在含有比金鱼（宿主）DNA多1000倍的混合物中，来自寄生虫丹尼列夫斯基锥虫的DNA会被优先扩增。此外，在存在寄生虫（血卵涡鞭虫属）自然感染的情况下，未感染宿主（坦氏雪蟹）的SSU rDNA没有出现微弱扩增。在来自感染的坦氏雪蟹的样本中，只有血卵涡鞭虫属的SSU rDNA被扩增。这种UNonMet-PCR是一种强大的工具，可用于从尚未从后生动物宿主中分离出来的非后生动物病原体或共生体中扩增SSU rDNA。
