Allan-Blitz Lao-Tzu, Adams Gordon, Sanders Gabriela, Shah Palak, Ramesh Krithik, Jarolimova Jana, Ard Kevin L, Branda John A, Klausner Jeffrey D, Sabeti Pardis C, Lemieux Jacob E
Division of Global Health Equity, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Broad Institute of Massachusetts Institute of Technology and Harvard, Boston, Massachusetts, USA.
mSphere. 2025 Jan 28;10(1):e0067724. doi: 10.1128/msphere.00677-24. Epub 2024 Dec 17.
Nucleic acid amplification testing (NAAT) for is unavailable in resource-limited settings. We previously developed a CRISPR-based lateral flow assay for detecting . We aimed to pair that assay with point-of-care DNA extraction, assess performance in clinical urine specimens, and optimize assay kinetics. We collected urine specimens among men presenting with urethritis enrolling in a clinical trial at the Massachusetts General Hospital Sexual Health Clinic. We assessed the quantified DNA yield of detergent-based extractions with and without heat. We selected one detergent for extracting all specimens, paired with isothermal recombinase polymerase amplification for 90 minutes and lateral flow Cas13a detection, interpreted via pixel intensity analysis. We also trained a smartphone-based machine-learning model on 1,008 images to classify lateral flow results. We used the model to interpret lateral flow results from the clinical specimens. We also tested a modified amplification chemistry with a second forward primer lacking the T7-promoter to accelerate reaction kinetics. Extraction with 0.02% Triton X resulted in an average DNA yield of 2.6 × 10 copies/µL (SD ± 6.7 × 10). We treated 40 urine specimens ( = 12 positive) with 0.02% Triton X, and using quantified pixel intensity analysis, the Cas13a-based assay correctly classified all specimens (100% agreement; 95% CI 91.2%-100%). The machine-learning model correctly classified 45/45 strips in the validation data set and all 40 lateral flow strips from clinical specimens. Including the second forward primer reduced incubation time to 60 minutes. Using point-of-care DNA extraction, our Cas13a-based lateral flow assay demonstrated promising performance among clinical urine specimens.IMPORTANCEUsing a CRISPR-based assay we previously developed for detection, we developed new techniques to facilitate point-of-care use. We then demonstrated the promising performance of that assay in clinical specimens. Furthermore, we developed a smartphone-based machine learning application for assisting interpretation of lateral flow strip results. Such an assay has the potential to transform the care of sexually transmitted infections in low-resource settings where diagnostic tests are unavailable. A point-of-care pathogen-specific assay, paired with the connectivity offered by a smartphone application, can also support public health surveillance efforts in such areas.
在资源有限的环境中无法进行核酸扩增检测（NAAT）。我们之前开发了一种基于CRISPR的侧向流动分析法用于检测[具体检测对象未明确给出]。我们旨在将该分析法与即时护理DNA提取相结合，评估其在临床尿液样本中的性能，并优化分析动力学。我们在马萨诸塞州总医院性健康诊所参与一项临床试验的尿道炎男性患者中收集尿液样本。我们评估了有热和无热条件下基于去污剂提取的定量DNA产量。我们选择一种去污剂用于提取所有样本，与等温重组酶聚合酶扩增90分钟及侧向流动Cas13a检测相结合，通过像素强度分析进行解读。我们还在1008张图像上训练了一个基于智能手机的机器学习模型，以对侧向流动结果进行分类。我们使用该模型解读临床样本的侧向流动结果。我们还测试了一种改良的扩增化学方法，使用第二个缺少T7启动子的正向引物来加速反应动力学。用0.02% Triton X提取平均DNA产量为2.6×10拷贝/微升（标准差±6.7×10）。我们用0.02% Triton X处理了40份尿液样本（n = 12份阳性），并使用定量像素强度分析，基于Cas13a的分析法正确分类了所有样本（100%一致；95%置信区间91.2% - 100%）。机器学习模型在验证数据集中正确分类了45/45条试纸，以及临床样本中的所有40条侧向流动试纸。加入第二个正向引物将孵育时间缩短至60分钟。使用即时护理DNA提取，我们基于Cas13a的侧向流动[具体检测对象未明确给出]分析法在临床尿液样本中表现出了有前景的性能。重要性我们利用之前开发的基于CRISPR的分析法进行[具体检测对象未明确给出]检测，开发了新技术以促进即时护理应用。然后我们证明了该分析法在临床样本中的有前景的性能。此外，我们开发了一个基于智能手机的机器学习应用程序来辅助解读侧向流动试纸结果。这样一种分析法有潜力在缺乏诊断检测的低资源环境中改变性传播感染的护理方式。一种即时护理病原体特异性分析法，与智能手机应用程序提供的连通性相结合，也可以支持这些地区的公共卫生监测工作。
