A multiplexed, allele-specific recombinase polymerase amplification assay with lateral flow readout for sickle cell disease detection,Lab on a Chip

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A multiplexed, allele-specific recombinase polymerase amplification assay with lateral flow readout for sickle cell disease detection
Lab on a Chip ( IF 6.1 ) Pub Date : 2024-07-25 , DOI: 10.1039/d4lc00281d
Megan M Chang ₁ , Mary E Natoli ₁ , Alexis F Wilkinson ₁ , Venée N Tubman _{2,

3} , Gladstone E Airewele _{2,

3} , Rebecca R Richards-Kortum ₁

Affiliation

Isothermal nucleic acid amplification tests have the potential to improve disease diagnosis at the point of care, but it remains challenging to develop multiplexed tests that can detect ≥3 targets or to detect point mutations that may cause disease. These capabilities are critical to enabling informed clinical decision-making for many applications, such as sickle cell disease (SCD). To address this, we describe the development of a multiplexed allele-specific recombinase polymerase amplification (RPA) assay with lateral flow readout. We first characterize the specificity of RPA using primer design strategies employed in PCR to achieve point mutation detection, and demonstrate the utility of these strategies in achieving selective isothermal amplification and detection of genomic DNA encoding for the healthy β^A globin allele, or genomic DNA containing point mutations encoding for pathologic β^S and β^C globin alleles, which are responsible for most sickle cell disorders. We then optimize reaction conditions to achieve multiplexed amplification and identification of the three alleles in a single reaction. Finally, we perform a small pilot study with 20 extracted genomic DNA samples from SCD patients and healthy volunteers – of the 13 samples with valid results, the assay demonstrated 100% sensitivity and 100% specificity for detecting pathologic alleles, and an overall accuracy of 92.3% for genotype prediction. This multiplexed assay is rapid, minimally instrumented, and when combined with point-of-care sample preparation, could enable DNA-based diagnosis of SCD in low-resource settings. The strategies reported here could be applied to other challenges, such as detection of mutations that confer drug resistance.

中文翻译：

用于镰状细胞病检测的多重等位基因特异性重组酶聚合酶扩增测定，具有侧流读数

等温核酸扩增测试有可能改善护理点的疾病诊断，但开发能够检测 ≥3 个靶标或检测可能导致疾病的点突变的多重测试仍然具有挑战性。这些功能对于镰状细胞病 (SCD) 等许多应用的明智临床决策至关重要。为了解决这个问题，我们描述了具有侧流读数的多重等位基因特异性重组酶聚合酶扩增（RPA）测定的开发。我们首先使用 PCR 中采用的引物设计策略来表征 RPA 的特异性，以实现点突变检测，并证明这些策略在实现选择性等温扩增和检测编码健康 β ^A珠蛋白等位基因的基因组 DNA 或包含编码病理性 β ^S和 β ^C球蛋白等位基因的点突变，这是大多数镰状细胞病的原因。然后，我们优化反应条件，以在单个反应中实现多重扩增和三个等位基因的鉴定。最后，我们对从 SCD 患者和健康志愿者中提取的 20 个基因组 DNA 样本进行了一项小型试点研究，在 13 个具有有效结果的样本中，该测定法显示出检测病理等位基因的 100% 灵敏度和 100% 特异性，总体准确度为 92.3 % 用于基因型预测。这种多重检测速度快、仪器设备最少，并且与现场样本制备相结合，可以在资源匮乏的环境中实现基于 DNA 的 SCD 诊断。这里报告的策略可以应用于其他挑战，例如检测赋予耐药性的突变。

更新日期：2024-07-25

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