Rapid electrostatic DNA enrichment for sensitive detection of Trichomonas vaginalis in clinical urinary samples.
Rapid electrostatic DNA enrichment for sensitive detection of Trichomonas vaginalis in clinical urinary samples.
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DOI：
10.1039/c9ay02478f
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发表时间：
2020-02-28
期刊：
影响因子：
--
通讯作者：
Cabodi M
中科院分区：
文献类型：
--
作者：
Rosenbohm JM;Robson JM;Singh R;Lee R;Zhang JY;Klapperich CM;Pollock NR;Cabodi M
阴道毛滴虫（TV）估计是全球最常见的非病毒性传播感染，可导致盆腔炎，妊娠并发症，并增加获得和传播艾滋病毒的风险。一旦确诊，TV感染可以用口服抗生素治疗;然而，感染者通常没有症状，不寻求治疗。世卫组织和其他组织已经确定需要即时检测来扩大TV检测和筛查的可及性;理想的检测特征包括高灵敏度和特异性以及使用尿液作为样本类型的能力，而不是侵入性收集的拭子样本。在这里，我们报告了一个概念验证原型，用于快速静电富集尿样中的DNA，并证明了使用大量尿液来提高下游核酸扩增检测的灵敏度。我们开发了一种内部控制的嗜热解旋酶依赖性扩增（tHDA）测定与侧流免疫测定读数，并证明这种tHDA测定可以直接在我们的DNA捕获过滤器上进行。我们使用具有qPCR定量TV负载的临床尿液样品验证了我们的方法。使用62个临床尿液样本和一个简单的样品处理装置，我们的tHDA检测显示96.6%的灵敏度和100%的特异性。当检测1 mL样品时，我们的分析检测限约为7个基因组当量的TV DNA/mL样品，与现有的TV等温检测相当。使用大体积模拟样本（40 mL加标TV DNA的缓冲尿液），我们还证明灵敏度可以提高28倍，达到每mL 0.25个基因组当量的TV DNA，样本处理时间仅为2分钟。
Estimated to be the most common non-viral sexually transmitted infection globally, Trichomonas vaginalis (TV) can lead to pelvic inflammatory disease, pregnancy complications, and increased risk of acquiring and transmitting HIV. Once diagnosed, TV infection can be treated with oral antibiotics; however, infected individuals are often asymptomatic and do not seek treatment. The WHO and others have identified a need for point-of-care tests to expand access to TV testing and screening; ideal test characteristics include high sensitivity and specificity and the ability to use urine as a sample type, rather than invasively collected swab samples. Here, we report on a proof-of-concept prototype for rapid, electrostatic enrichment of DNA from urine samples and demonstrate the use of large volumes of urine to increase sensitivity of downstream nucleic acid amplification testing. We developed an internally controlled thermophilic helicase-dependent amplification (tHDA) assay with lateral flow immunoassay readout and demonstrate that this tHDA assay can be performed directly on our DNA capture filter. We validated our method using clinical urine samples with qPCR-quantified TV loads. Using 62 clinical urine samples and a simple sample processing device, our tHDA assay displayed 96.6% sensitivity and 100% specificity. Our analytical limit of detection was found to be approximately 7 genomic equivalents of TV DNA per mL of sample when 1 mL of sample was tested, comparable to existing isothermal tests for TV. Using large-volume simulated samples (40 mL of buffered urine with spiked-in TV DNA), we also demonstrated that sensitivity could be improved 28-fold to 0.25 genomic equivalents of TV DNA per mL, with a sample processing time of only 2 minutes.
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