Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, U. S. Food and Drug Administration, Bethesda, Maryland, United States of America.
PLoS One. 2012;7(8):e43533. doi: 10.1371/journal.pone.0043533. Epub 2012 Aug 22.
Trypanosoma cruzi, a blood-borne parasite, is the etiological agent of Chagas disease. T. cruzi trypomastigotes, the infectious life cycle stage, can be detected in blood of infected individuals using PCR-based methods. However, soon after a natural infection, or during the chronic phase of Chagas disease, the number of parasites in blood may be very low and thus difficult to detect by PCR. To facilitate PCR-based detection methods, a parasite concentration approach was explored. A whole cell SELEX strategy was utilized to develop serum stable RNA aptamers that bind to live T. cruzi trypomastigotes. These aptamers bound to the parasite with high affinities (8-25 nM range). The highest affinity aptamer, Apt68, also demonstrated high specificity as it did not interact with the insect stage epimastigotes of T. cruzi nor with other related trypanosomatid parasites, L. donovani and T. brucei, suggesting that the target of Apt68 was expressed only on T. cruzi trypomastigotes. Biotinylated Apt68, immobilized on a solid phase, was able to capture live parasites. These captured parasites were visible microscopically, as large motile aggregates, formed when the aptamer coated paramagnetic beads bound to the surface of the trypomastigotes. Additionally, Apt68 was also able to capture and aggregate trypomastigotes from several isolates of the two major genotypes of the parasite. Using a magnet, these parasite-bead aggregates could be purified from parasite-spiked whole blood samples, even at concentrations as low as 5 parasites in 15 ml of whole blood, as detected by a real-time PCR assay. Our results show that aptamers can be used as pathogen specific ligands to capture and facilitate PCR-based detection of T. cruzi in blood.
克氏锥虫是一种血液传播的寄生虫，是恰加斯病的病原体。锥虫的循环型感染阶段——锥鞭毛体，可以通过基于 PCR 的方法在感染个体的血液中检测到。然而，在自然感染后不久，或恰加斯病的慢性期，血液中的寄生虫数量可能非常低，因此很难通过 PCR 检测到。为了促进基于 PCR 的检测方法，探索了一种寄生虫浓缩方法。利用全细胞 SELEX 策略，开发了与活克氏锥虫锥鞭毛体结合的血清稳定 RNA 适体。这些适体与寄生虫具有高亲和力（8-25 nM 范围）。亲和力最高的适体 Apt68 也表现出高度特异性，因为它与昆虫阶段的前鞭毛体的 T. cruzi 或其他相关的利什曼原虫寄生虫，L. donovani 和 T. brucei 均无相互作用，这表明 Apt68 的靶标仅在 T. cruzi 锥鞭毛体上表达。固定在固相上的生物素化 Apt68 能够捕获活寄生虫。当适体包被的顺磁珠与锥鞭毛体表面结合时，这些被捕获的寄生虫在显微镜下可见，形成大的运动聚集物。此外，Apt68 还能够从寄生虫的两个主要基因型的几种分离株中捕获和聚集锥鞭毛体。使用磁铁，可以从寄生虫感染的全血样本中，甚至在全血中低至 5 个寄生虫的浓度下，通过实时 PCR 检测，从寄生虫感染的全血样本中纯化这些寄生虫-珠粒聚集物。我们的结果表明，适体可用作病原体特异性配体，用于捕获和促进血液中 T. cruzi 的基于 PCR 的检测。
