Allele-Specific Recombinase Polymerase Amplification to Detect Sickle Cell Disease in Low-Resource Settings.
Allele-Specific Recombinase Polymerase Amplification to Detect Sickle Cell Disease in Low-Resource Settings.
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等位基因特异性重组酶聚合酶扩增检测资源匮乏环境中的镰刀细胞病
DOI：
10.1021/acs.analchem.0c04191
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发表时间：
2021-03-23
影响因子：
7.4
通讯作者：
Richards-Kortum RR
中科院分区：
文献类型：
--
作者：
Natoli ME;Chang MM;Kundrod KA;Coole JB;Airewele GE;Tubman VN;Richards-Kortum RR
镰状细胞病（SCD）是一组常见的、危及生命的疾病，由β珠蛋白基因的点突变引起。众所周知，通过新生儿和幼儿筛查、父母教育和预防性治疗进行早期诊断可以降低死亡率。然而，传统诊断方法的成本和复杂性限制了在全球资源有限地区早期诊断SCD的可行性。虽然有几种即时检测是商业上可用的，但大多数是基于抗体的检测，不能用于最近接受输血的患者。在这里，我们描述了一种快速，低成本的核酸检测，使用实时荧光检测点突变编码血红蛋白S（HbS）在一轮等温重组酶聚合酶扩增（RPA）的发展。当用来自SCD患者和健康志愿者的一组临床样本进行测试时，我们的检测方法对βA珠蛋白和βS珠蛋白等位基因的灵敏度均为100%，对βA珠蛋白等位基因和βS珠蛋白等位基因的特异性分别为94.7%和97.1%（n = 91）。最后，我们证明了概念验证的样品回答基因分型的基因组DNA从毛细血管血使用碱裂解程序和稀释的裂解液直接输入到RPA。该工作流程在<30分钟内以<5美元的成本在市售台式荧光计和开源微型荧光计上进行。这项研究证明了一种用于SCD的快速，样本到答案的核酸检测的潜在效用，该检测可以在护理点附近实施，并且可以适用于基因组DNA中的其他致病点突变。
Sickle cell disease (SCD) is a group of common, life-threatening disorders caused by a point mutation in the β globin gene. Early diagnosis through newborn and early childhood screening, parental education, and preventive treatments are known to reduce mortality. However, the cost and complexity of conventional diagnostic methods limit the feasibility of early diagnosis for SCD in resource-limited areas worldwide. Although several point-of-care tests are commercially available, most are antibody-based tests, which cannot be used in patients who have recently received a blood transfusion. Here, we describe the development of a rapid, low-cost nucleic acid test that uses real-time fluorescence to detect the point mutation encoding hemoglobin S (HbS) in one round of isothermal recombinase polymerase amplification (RPA). When tested with a set of clinical samples from SCD patients and healthy volunteers, our assay demonstrated 100% sensitivity for both the βA globin and βS globin alleles and 94.7 and 97.1% specificities for the βA globin allele and βS globin allele, respectively (n = 91). Finally, we demonstrate proof-of-concept sample-to-answer genotyping of genomic DNA from capillary blood using an alkaline lysis procedure and direct input of diluted lysate into RPA. The workflow is performed in <30 min at a cost of <$5 USD on a commercially available benchtop fluorimeter and an open-source miniature fluorimeter. This study demonstrates the potential utility of a rapid, sample-to-answer nucleic acid test for SCD that may be implemented near the point of care and could be adapted to other disease-causing point mutations in genomic DNA.
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影响因子：
7.4
作者：
Ng, Benjamin Y. C.;Wee, Eugene J. H.;Trau, Matt
通讯作者：
Trau, Matt
影响因子：
5.1
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Vichinsky, EP
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Boehringer, K. F.
影响因子：
3
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通讯作者：
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影响因子：
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