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Evaluation of targeted next-generation sequencing for detection of equine pathogens in clinical samples
The Journal of Veterinary Diagnostic Investigation ( IF 1.5 ) Pub Date : 2020-12-11 , DOI: 10.1177/1040638720978381
Eman Anis 1, 2 , Marcia R S Ilha 3 , Julie B Engiles 1 , Rebecca P Wilkes 3, 4
Affiliation  

Equine infectious disease outbreaks may have profound economic impact, resulting in losses of millions of dollars of revenue as a result of horse loss, quarantine, and cancelled events. Early and accurate diagnosis is essential to limit the spread of infectious diseases. However, laboratory detection of infectious agents, especially the simultaneous detection of multiple agents, can be challenging to the clinician and diagnostic laboratory. Next-generation sequencing (NGS), which allows millions of DNA templates to be sequenced simultaneously in a single reaction, is an ideal technology for comprehensive testing. We conducted a proof-of-concept study of targeted NGS to detect 62 common equine bacterial, viral, and parasitic pathogens in clinical samples. We designed 264 primers and constructed a bioinformatics tool for the detection of targeted pathogens. The designed primers were able to specifically detect the intended pathogens. Results of testing 27 clinical samples with our targeted NGS assay compared with results of routine tests (assessed as a group) yielded positive percent agreement of 81% and negative percent agreement of 83%, overall agreement of 81%, and kappa of 0.56 (moderate agreement). This moderate agreement was likely the result of low sensitivity of some primers. However, our NGS assay successfully detected multiple pathogens in the clinical samples, including some pathogens missed by routine techniques.



中文翻译:

评估用于检测临床样本中马病原体的靶向二代测序

马传染病爆发可能会产生深远的经济影响,由于马匹损失、检疫和活动取消,导致数百万美元的收入损失。早期准确的诊断对于限制传染病的传播至关重要。然而,传染性病原体的实验室检测,尤其是同时检测多种病原体,对临床医生和诊断实验室来说可能具有挑战性。新一代测序 (NGS) 允许在一次反应中同时对数百万个 DNA 模板进行测序,是进行全面测试的理想技术。我们对靶向 NGS 进行了概念验证研究,以检测临床样本中的 62 种常见马细菌、病毒和寄生虫病原体。我们设计了264个引物并构建了一个用于检测目标病原体的生物信息学工具。设计的引物能够特异性检测预期的病原体。使用我们的靶向 NGS 分析测试 27 个临床样本的结果与常规测试的结果(作为一个组评估)产生了 81% 的阳性一致性百分比和 83% 的阴性一致性百分比、81% 的总体一致性和 0.56 的 kappa(中等协议)。这种适度的一致性可能是某些引物灵敏度低的结果。然而,我们的 NGS 检测成功地检测到临床样本中的多种病原体,包括一些常规技术遗漏的病原体。使用我们的靶向 NGS 分析测试 27 个临床样本的结果与常规测试的结果(作为一个组评估)产生了 81% 的阳性一致性百分比和 83% 的阴性一致性百分比、81% 的总体一致性和 0.56 的 kappa(中等协议)。这种适度的一致性可能是某些引物灵敏度低的结果。然而,我们的 NGS 检测成功地检测到临床样本中的多种病原体,包括一些常规技术遗漏的病原体。使用我们的靶向 NGS 分析测试 27 个临床样本的结果与常规测试的结果(作为一个组评估)产生了 81% 的阳性一致性百分比和 83% 的阴性一致性百分比、81% 的总体一致性和 0.56 的 kappa(中等协议)。这种适度的一致性可能是某些引物灵敏度低的结果。然而,我们的 NGS 检测成功地检测到临床样本中的多种病原体,包括一些常规技术遗漏的病原体。

更新日期:2020-12-11
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