SIMPLseq:一个高灵敏度的Plasmodium falciparum基因型和PCR污染跟踪工具
Philipp Schwabl1,2,3, Jorge-Eduardo Amaya-Romero1,2, Katrina A Kelley1,2
1Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA.
一个新的Plasmodium falciparum amplicon测序 (AmpSeq) 迷你复合器,SIMPLseq,提供高灵敏度的疟疾寄生虫基因定型和综合污染检测. 该工具增强了基因组流行病学,用于药物耐药性跟踪和干预试验.
科学领域:
- 基因组流行病学基因组流行病学
- 分子诊断学 分子诊断学
- 寄生虫学的寄生虫学
背景情况:
- 聚合酶连锁反应 (PCR) 片序列 (AmpSeq) 对于病原体基因定型和疾病监测至关重要.
- 现有的大型AmpSeq面板与低寄生病样本 (<10寄生虫/μl) 斗争,限制了它们在疟疾药物耐药性跟踪中的使用.
- 管理PCR污染是AmpSeq方法的重大挑战.
研究的目的:
- 开发一种高灵敏度的Plasmodium falciparum AmpSeq miniplex (SIMPLseq),用于低寄生病样本.
- 将污染检测系统集成到迷你综合体中,使用内联条形码.
- 为了评估SIMPLseq的灵敏度,精度和污染检测能力.
主要方法:
- 开发了一个6位 Plasmodium falciparum AmpSeq迷你复合体 (SIMPLseq),用于第一轮PCR (PCR1) 污染检测.
- 通过使用全基因组测序 (WGS) 数据在基板中评估多样性.
- 使用模拟样本来比较SIMPLseq的灵敏度和精度与4CAST迷你复合器.
- 蓄意引入污染事件以评估检测严格性和估计来自马里样本的意外污染率.
主要成果:
- SIMPLseq 显示出高的 in silico 类型多样性,区分了 96.0% 的样本对.
- 在≥0.5寄生虫/μl和≥50%在0.125寄生虫/μl时实现了100%的平均位点检测,表现优于4CAST.
- 在线条形码没有显著影响产量,并且正确识别了所有24个故意引入的PCR1污染.
- 在来自马里的1420个样本中,确定了39个无意的污染.
结论:
- 通过提供高灵敏度的P. falciparum基因型识别和PCR污染检测,SIMPLseq增强了疟疾基因组流行病学.
- 该协议很简单,使用开源试剂,避免昂贵的预放大.
- SIMPLseq对于分类复发性感染,估计多克隆性和在试验中应用基因型感染终点非常有价值.
更多相关视频
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