Preferential PCR amplification of parasitic protistan small subunit rDNA from metazoan tissues
Preferential PCR amplification of parasitic protistan small subunit rDNA from metazoan tissues
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DOI：
10.1111/j.1550-7408.2004.tb00574.x
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发表时间：
2004-05-01
影响因子：
2.2
通讯作者：
Mak, MWS
中科院分区：
文献类型：
--
作者：
Bower, SM;Carnegie, RB;Mak, MWS
验证了一种选择性扩增一段非后生动物小亚基(SSU)rDNA基因的“通用非后生动物”聚合酶链式反应(U NonMet-PCR)。所用的引物为：18S-EUK581-F(5‘-GTGCCAGCAGCCGCG-3’)和18S-EUK1134-R(5‘-TTTAAGTTTCAGCCTTGCG-3’)。它的靶点在古生物、细菌和真核生物(包括真菌)中高度保守，但不是大多数后生动物(除了孔虫目、鞭毛目和粘虫)在第14和19碱基上存在错配，导致扩增不佳或失败。在验证过程中，UNonMet-PCR从所有被检测的原生动物(7个较高的类群中有16个，包括两种海洋浮游植物)和真菌(3个)中扩增出SSU rDNA基因片段，但从大多数被检测的后生动物中扩增得很少或根本没有(从8个较高的类群中扩增13个)。当后生动物宿主中存在非后生动物寄生虫时，寄生虫DNA优先被扩增。例如，寄生虫达尼列夫斯基锥虫的DNA在含有高达1000倍的金鱼鲫鱼(宿主)DNA的混合物中被优先扩增。此外，未感染的宿主(丹尼氏绒毛虫)SSU rDNA在自然感染寄生虫(Hematoddium sp.)的情况下也不会发生微弱的扩增。只有血藻属(hematoddium sp.)从感染丹宁革兰氏杆菌的样本中扩增出SSU rDNA。这种UNonMet-PCR也是一种从非后生动物病原体或共生体中扩增SSU rDNA的强大方法。
A "universal non-metazoan" polymerase chain reaction (U NonMet-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 1,000X 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 too] for amplifying SSU rDNA from non-metazoan pathogens or symbionts that have not been isolated from metazoan hosts.