PANDAA intentionally violates conventional qPCR design to enable durable, mismatch-agnostic detection of highly polymorphic pathogens.
PANDAA intentionally violates conventional qPCR design to enable durable, mismatch-agnostic detection of highly polymorphic pathogens.
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DOI：
10.1038/s42003-021-01751-9
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发表时间：
2021-02-18
影响因子：
5.9
通讯作者：
Essex M
中科院分区：
文献类型：
--
作者：
MacLeod IJ;Rowley CF;Essex M
敏感和可重复的诊断是遏制现有和新出现的病原体传播的基础。尽管临床病毒学依赖于qPCR，但技术挑战仍然存在，因为探针结合区域的序列不稳定性会产生假阴性结果，从而损害其可持续流行病遏制的可靠性。我们系统地违反了规范的qPCR设计原则，开发了泛简并扩增和适应（PANDAA），这是一种点突变试验，可减轻序列变异对基于探针的qPCR性能的影响。以HIV-1为模型系统，我们优化并验证了PANDAA检测HIV耐药突变（DRMs）的方法。具有与探针结合位点重叠的3'末端的超简并引物在qPCR期间通过定点诱变来适应靶标，以取代DRM近端序列变异。PANDAA定量DRM的出现频率≥5%（从核酸到结果的2小时），灵敏度和特异性分别为96.9%和97.5%。PANDAA是一种创新性的进步，适用于任何病原体，其中靶近端遗传变异性阻碍了诊断发展。Iain麦克劳德等人开发了泛简并扩增和适应（PANDAA）点突变试验，用于检测HIV耐药突变。这种方法可以应用于任何病原体的目标近端遗传变异性阻碍诊断的发展。
Sensitive and reproducible diagnostics are fundamental to containing the spread of existing and emerging pathogens. Despite the reliance of clinical virology on qPCR, technical challenges persist that compromise their reliability for sustainable epidemic containment as sequence instability in probe-binding regions produces false-negative results. We systematically violated canonical qPCR design principles to develop a Pan-Degenerate Amplification and Adaptation (PANDAA), a point mutation assay that mitigates the impact of sequence variation on probe-based qPCR performance. Using HIV-1 as a model system, we optimized and validated PANDAA to detect HIV drug resistance mutations (DRMs). Ultra-degenerate primers with 3’ termini overlapping the probe-binding site adapt the target through site-directed mutagenesis during qPCR to replace DRM-proximal sequence variation. PANDAA-quantified DRMs present at frequency ≥5% (2 h from nucleic acid to result) with a sensitivity and specificity of 96.9% and 97.5%, respectively. PANDAA is an innovative advancement with applicability to any pathogen where target-proximal genetic variability hinders diagnostic development. Iain MacLeod et al. develop the Pan-Degenerate Amplification and Adaptation (PANDAA) point mutation assay to detect HIV drug resistance mutations. This method can be applied to any pathogen where target-proximal genetic variability hinders diagnostic development.
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