DETECTION OF TRYPANOSOMA-CONGOLENSE AND TRYPANOSOMA-BRUCEI SUBSPECIES BY DNA AMPLIFICATION USING THE POLYMERASE CHAIN-REACTION
DETECTION OF TRYPANOSOMA-CONGOLENSE AND TRYPANOSOMA-BRUCEI SUBSPECIES BY DNA AMPLIFICATION USING THE POLYMERASE CHAIN-REACTION
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DOI：
10.1017/s0031182000061023
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发表时间：
1989-08-01
期刊：
影响因子：
2.4
通讯作者：
DONELSON, JE
中科院分区：
文献类型：
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作者：
MOSER, DR;COOK, GA;DONELSON, JE
刚果锥虫的核 DNA 包含一系列高度保守的 369 个碱基对 (bp) 重复序列。确定了这些重复的三个克隆拷贝的序列。先前已证明布氏锥虫的核 DNA 中存在一个不相关的 177 bp 重复家族（Sloof 等，1983 a）。合成了寡核苷酸，通过聚合酶链式反应 (PCR) 启动这些重复 DNA 的特异性扩增。刚果锥虫或布氏锥虫单一寄生虫中 10% 的 DNA 扩增。产生了足够的扩增产物，在用溴化乙锭染色的琼脂糖凝胶中可见条带。这种检测水平不依赖于放射性的使用，比以前基于放射性 DNA 探针的检测方法灵敏约 100 倍。这些寡核苷酸不会引发其他锥虫物种、利什曼原虫、小鼠或人类 DNA 中 DNA 序列的扩增。 感染刚果锥虫和/或布氏锥虫的动物血液中 DNA 的扩增。允许鉴定远低于显微镜检查可检测到的寄生虫水平。由于 PCR 扩增可以同时对大量样品进行，因此非常适合对哺乳动物血液和昆虫媒介中非洲锥虫的流行情况进行大规模研究。
The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983 a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.