通过检测过滤人尿样本中的物种特异性重复基因组 DNA 诊断南美锥虫病

Miriam Price, Nilanjan Lodh
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摘要

导言:在美洲发现的由克鲁斯锥虫(T cruzi)引起的恰加斯病,由于缺乏灵敏的诊断检测,在感染的早期阶段常常被漏诊。在急性期,由于感染的非特异性和低抗体水平,以及在慢性期,由于血液中锥虫水平较低,传统的免疫学和寄生虫学检测常常失效。为了成功实施控制策略,必须有一种敏感而特异的诊断检测方法:我们通过以下方法证明了通过聚合酶链式反应(PCR)检测克鲁兹锥虫特异性重复 DNA 的可能性(概念验证):(1) 在 15 份从没有感染过克鲁兹锥虫(3 株)、布氏锥虫和罗得西亚锥虫(非洲株)的志愿者尿液样本中添加 3 种不同浓度的克鲁兹锥虫基因组 DNA;(2) 从过滤收集的阿根廷临床样本中检测克鲁兹锥虫特异性重复 DNA。使用了三组引物:结果:我们的方法通过两组引物从 1 份临床样本中检测到了特异性克鲁兹锥虫菌株的重复 DNA,通过所有 3 组引物从加标尿液中检测到了特异性克鲁兹锥虫菌株的重复 DNA,但没有检测到非洲菌株。我们还对 T cruzi 菌株进行了序列稀释(加标),以检测检测方法的灵敏度。其中一组引物可持续检测到所有克鲁兹绦虫菌株的卫星 DNA,检测范围从 70 pg/μl 到 175 fg/μl:我们通过灵敏而特异的 PCR 分析证明了从过滤尿液样本中检测 T cruzi 特异性 DNA 的可行性。除了引物的灵敏度和特异性明显提高外,我们的方法还可用于检测恰加斯病流行地区的恰加斯病患病率--尤其是先天性恰加斯病新生儿筛查--以及急性期恰加斯病患病率。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Diagnosis of Chagas Disease by Detecting Species-Specific Repetitive Genomic DNA from Filtered Human Urine Samples.

Introduction: Chagas disease caused by Trypanosoma cruzi (T cruzi) found in the Americas is often missed during the early stage of infection due to lack of sensitive diagnostic tests. The classic immunological and parasitological tests often fail in the acute phase due to the nonspecific and low antibody level nature of the infection and in the chronic phase due to low levels of trypanosomes in the blood. For successful control strategies, there must be a sensitive and specific diagnostic test.

Objective/methods: We have demonstrated the possibility (proof of concept) of detecting T cruzi-specific repeat DNA via polymerase chain reaction (PCR) by (1) spiking 15 urine samples collected from volunteers free of prior infection with 3 different concentrations of T cruzi (3 strains), Trypanosoma brucei, and Trypanosoma rhodesiense (African strain) genomic DNA and (2) from filtered collected clinical samples from Argentina. Three sets of primers were used.

Results: Our approach detected repeat DNA specific for T cruzi strains from 1 clinical sample by 2 sets of primer and from spiked urine by all 3 sets of primer but not the African species. A serial dilution (spiking) also was performed on T cruzi strains to detect sensitivities of the assay. One set of primers constantly detected satellite DNA for all T cruzi strains from 70 pg/μl to 175 fg/μl.

Conclusions: We were able to demonstrate the feasibility of detecting T cruzi-specific DNA from filtered urine samples by sensitive and specific PCR assay. Besides the evident increased sensitivity and specificity of primers, our approach can be used to explore Chagas prevalence in endemic areas - especially in congenital Chagas newborn screening - and in the acute phase.

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