Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.
Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.
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使用新型快速等温扩增侧流测定法检测三日疟原虫。
DOI：
10.1101/2023.02.26.23286371
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Parr,JonathanB
中科院分区：
文献类型：
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作者：
Assefa,Ashenafi;Wamae,KevinK;Hennelly,ChrisM;Ngasala,Billy;Muller,Meredith;Kalonji,Albert;Phanzu,Fernandine;Cunningham,ClarkH;Lin,JessicaT;Parr,JonathanB
背景恶性疟和间日疟是疟疾发病和死亡的主要原因，疟疾和其他疟原虫的感染也会导致发病和死亡。目前对这些感染的了解在一定程度上受到现有的医疗点诊断方法的限制，这些方法未能区分它们，并且对低密度感染的敏感度很低。目前，准确的诊断需要在资源充足的实验室进行分子分析。这份报告描述了AP的发展。采用快速、等温重组酶聚合酶扩增(RPA)和侧向流纸条检测的疟疾诊断方法。疟疾基因组序列，并通过修改已公布的引物组。根据制造商RPA反应条件(TwistDx NFO试剂盒)，优化了针对多拷贝P的等温分析。疟疾18S rRNA基因，孵育温度39℃，运行时间30分钟。使用侧条(用夹心免疫分析法检测到的FAM标记的生物素化扩增子，使用金纳米颗粒显示)显示RPA产物。结果用18S rRNA质粒DNA检测的灵敏度为10拷贝/µL(~ 1.7基因组等价物)，分析特异性为100%。现场样本检测显示，与qPCR相比，临床敏感性为95%，特异性为88%。结论与简化的DNA提取方法相结合，该方法有可能成为未来现场部署、护理点使用的检测方法。疟疾感染，这在很大程度上仍未得到诊断，但却是慢性疟疾的一个被忽视的原因。该分析在横向流动条上提供快速、简单的读数，而不需要昂贵的实验室设备。
BackgroundWhilePlasmodium falciparumandPlasmodium vivaxcause the majority of malaria cases and deaths, infection byPlasmodium malariaeand otherPlasmodiumspecies also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of aP. malariaediagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection.MethodsMultiple combinations of custom RPA primers and probes were designed using publicly availableP. malariaegenomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopyP. malariae18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo.ResultsUsing 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min.ConclusionCombined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detectP. malariaeinfection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.