PLASMODIUM - GENUS-CONSERVED PRIMERS FOR SPECIES IDENTIFICATION AND QUANTITATION
PLASMODIUM - GENUS-CONSERVED PRIMERS FOR SPECIES IDENTIFICATION AND QUANTITATION
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DOI：
10.1006/expr.1995.1107
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发表时间：
1995-09-01
影响因子：
2.1
通讯作者：
MCCUTCHAN, TF
中科院分区：
文献类型：
--
作者：
LI, J;WIRTZ, RA;MCCUTCHAN, TF
稳定的RNA具有初级序列区域，这些区域在疟原虫属的每个成员中几乎相同，在宿主或其他常见病原体中没有发现。在疟原虫核糖体小亚基RNA中发现了几个‘属保守’序列，它们位于高变区的两侧。基于这些保守序列的引物允许从未知组成的样本中扩增物种-甚至可能是菌株特有的序列。作为这种方法的一个例子，从Giemsa染色的血液涂片中成功地恢复了四种人类疟疾物种的序列，包括卵形疟原虫的两个不同序列(相似性为91.5%)。这类信息对任何疟疾物种的流行病学和系统发育分析都很有用。我们发现，在聚合酶扩增周期中，rRNA衍生序列的扩增是以竞争的方式进行的，因此来自疟原虫物种的靶序列被扩增的比例与它们在样本中的丰度成比例。这一发现有几个含义。(1)混合感染标本扩增不同产物的比例与感染物种的比例密切相关。(2)当RT/PCR反应中加入已知数量的竞争RNA时，可以直接定量检测样本中的寄生虫核酸。(3)使用种属特异的引物扩增rRNA序列，使人们能够监测蚊子体内寄生虫的发育。(C)1995年学术出版社。
Stable RNAs have regions of primary sequence that are nearly identical in every member of the Plasmodium genus and not found in the host or in other common pathogens. Several ''genus-conserved'' sequences, which flank hypervariable regions, were identified within the small subunit ribosomal RNA of Plasmodium species. Primers based on these conserved sequences permit amplification of species- or possibly even strain-specific sequences from samples of unknown composition. As an example of this approach, sequences from the four human malaria species were successfully recovered from Giemsa-stained blood smears, including two different sequences for Plasmodium ovale (of 91.5% similarity). This type of information is useful for epidemiological and phylogenetic analysis of any malaria species. We show that amplification of rRNA-derived sequences behaves in a competitive fashion during the cycles of polymerase amplification and therefore target sequences from Plasmodium species are amplified in proportion to their abundance in the sample. There are several implications of this finding. (1) The proportion of different products resulting from amplification from samples with mixed infections is closely related to the proportion of infecting species, (2) Direct quantitation of parasite nucleic acids within a sample can be derived when known amounts of competitor RNA are added to the RT/PCR reaction. (3) Amplification of rRNA sequences, using genus-specific primers, allows one to monitor the development of the parasite in the mosquito. (C) 1995 Academic Press, Inc.