DETECTION OF TRYPANOSOMA-CRUZI BY DNA AMPLIFICATION USING THE POLYMERASE CHAIN-REACTION
DETECTION OF TRYPANOSOMA-CRUZI BY DNA AMPLIFICATION USING THE POLYMERASE CHAIN-REACTION
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DOI：
10.1128/jcm.27.7.1477-1482.1989
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发表时间：
1989-07-01
影响因子：
9.4
通讯作者：
DONELSON, JE
中科院分区：
文献类型：
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作者：
MOSER, DR;KIRCHHOFF, LV;DONELSON, JE
聚合酶链反应被用来扩增一个188碱基对（bp）的重复195 bp的克氏锥虫的核DNA序列，这是最丰富的序列在这种生物体的片段。该反应在四个T中扩增了该重复元件。Cruzi分离物来自广泛分离的地理区域。当从利什曼原虫属提取DNA时，使用了非洲锥虫或来自小鼠和人类的血液样本。从单个T.克氏寄生虫产生了一定量的188-bp元件，其在用溴化乙锭染色的凝胶中很容易看到。放射性标记的探针与膜结合的扩增产物的杂交将灵敏度提高到可以检测到单个寄生虫中1/200的DNA的水平。t.从实验室饲养的受感染昆虫媒介的腹部内容物中提取的DNA中很容易检测到cruzi DNA。在两名已知感染T的人的血液样本中没有检测到寄生虫DNA。cruzi，可能是因为在这些患者中循环寄生虫的数量极低，或者因为寄生虫血症是间歇性的。这些结果代表了一个相当大的增加灵敏度比以前报道的方法检测T。克鲁兹感染。聚合酶链反应扩增可用于在一天内评价大量样本，因此在大规模T。cruzi在昆虫媒介和哺乳动物宿主中。
The polymerase chain reaction was used to amplify a 188-base pair (bp) segment of the repetitive 195-bp nuclear DNA sequence of Trypanosoma cruzi that is the most abundant sequence in this organism. The reaction amplified this repetitive element in four T. cruzi isolates from widely separated geographic regions. No amplification of the 188-bp fragment occurred when DNAs extracted from Leishmania spp., African trypanosomes, or blood samples from mice and humans were used. Amplification of one-half of the DNA from a single T. cruzi parasite produced an amount of the 188-bp element that was readily visible in a gel stained with ethidium bromide. Hybridization of a radiolabeled probe to membrane-bound amplification products increased the sensitivity to a level at which 1/200 of the DNA in a single parasite could be detected. T. cruzi DNA was readily detected in DNA extracted from the abdominal contents of infected insect vectors reared in the laboratory. No parasite DNA was detected in the blood samples of two individuals known to be infected with T. cruzi, possibly because in such patients the numbers of circulating parasites are extremely low or because parasitemias are intermittent. These results represent a considerable increase in sensitivity over previously reported methods for the detection of T. cruzi infections. Polymerase chain reaction amplification can be used to evaluate large numbers of samples in a single day and thus should be useful in large-scale studies of the prevalence of T. cruzi in both insect vectors and mammalian hosts.