通过选择性病原体 DNA 富集和深度扩增子测序对血液寄生虫进行敏感的通用检测。

Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing.

机构信息

Parasitic Diseases Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, GA, USA.

Oak Ridge Institute for Science and Education, Oak Ridge, TN, USA.

出版信息

Microbiome. 2021 Jan 2;9(1):1. doi: 10.1186/s40168-020-00939-1.

DOI:10.1186/s40168-020-00939-1

PMID:33388088

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7778815/

Abstract

BACKGROUND

Targeted amplicon deep sequencing (TADS) has enabled characterization of diverse bacterial communities, yet the application of TADS to communities of parasites has been relatively slow to advance. The greatest obstacle to this has been the genetic diversity of parasitic agents, which include helminths, protozoa, arthropods, and some acanthocephalans. Meanwhile, universal amplification of conserved loci from all parasites without amplifying host DNA has proven challenging. Pan-eukaryotic PCRs preferentially amplify the more abundant host DNA, obscuring parasite-derived reads following TADS. Flaherty et al. (2018) described a pan-parasitic TADS method involving amplification of eukaryotic 18S rDNA regions possessing restriction sites only in vertebrates. Using this method, host DNA in total DNA extracts could be selectively digested prior to PCR using restriction enzymes, thereby increasing the number of parasite-derived reads obtained following NGS. This approach showed promise though was only as sensitive as conventional PCR.

RESULTS

Here, we expand on this work by designing a second set of pan-eukaryotic primers flanking the priming sites already described, enabling nested PCR amplification of the established 18S rDNA target. This nested approach facilitated introduction of a second restriction digestion between the first and second PCR, reducing the proportional mass of amplifiable host-derived DNA while increasing the number of PCR amplification cycles. We applied this method to blood specimens containing Babesia, Plasmodium, various kinetoplastids, and filarial nematodes and confirmed its limit of detection (LOD) to be approximately 10-fold lower than previously described, falling within the range of most qPCR methods.

CONCLUSIONS

The assay detects and differentiates the major malaria parasites of humans, along with several other clinically important blood parasites. This represents an important step towards a TADS-based universal parasite diagnostic (UPDx) test with a sufficient LOD for routine applications. Video Abstract.

摘要

背景

靶向扩增子深度测序（TADS）能够对不同的细菌群落进行特征描述，但将 TADS 应用于寄生虫群落的进展相对缓慢。最大的障碍是寄生虫制剂的遗传多样性，包括蠕虫、原生动物、节肢动物和一些棘头动物。与此同时，在不扩增宿主 DNA 的情况下，从所有寄生虫中普遍扩增保守基因座一直具有挑战性。泛真核 PCR 优先扩增更丰富的宿主 DNA，在 TADS 后掩盖寄生虫衍生的读数。Flaherty 等人。（2018 年）描述了一种涉及扩增仅在脊椎动物中具有限制性位点的真核 18S rDNA 区域的泛寄生虫 TADS 方法。使用这种方法，可以使用限制性内切酶在 PCR 之前选择性地消化总 DNA 提取物中的宿主 DNA，从而增加 NGS 后获得的寄生虫衍生读数的数量。尽管这种方法与传统 PCR 一样敏感，但显示出了希望。

结果

在这里，我们通过设计第二组围绕已经描述的引物位点的泛真核引物来扩展这项工作，从而能够对已建立的 18S rDNA 靶标进行嵌套 PCR 扩增。这种嵌套方法便于在第一和第二 PCR 之间引入第二次限制性消化，减少了可扩增宿主衍生 DNA 的比例质量，同时增加了 PCR 扩增循环的数量。我们将这种方法应用于含有巴贝虫、疟原虫、各种动基体目原生动物和丝虫的血液标本，并证实其检测限（LOD）比之前描述的低约 10 倍，落在大多数 qPCR 方法的范围内。