Allan-Blitz Lao-Tzu, Sanders Gabriela, Shah Palak, Adams Gordon, Jarolimova Jana, Ard Kevin, 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, MA.
Broad Institute of Massachusetts Institute of Technology and Harvard, Boston, MA.
medRxiv. 2024 Mar 4:2024.03.01.24303603. doi: 10.1101/2024.03.01.24303603.
Diagnosis of is dependent on nucleic acid amplification testing (NAAT), which is not available in resource-limited settings where the prevalence of infection is highest. Recent advances in molecular diagnostics leveraging the high specificity of CRISPR enzymes can permit field-deployable, point-of-care lateral flow assays. We previously reported on the development and performance of a lateral flow assay for detecting . Here we aimed to pair that assay with point-of-care DNA extraction techniques and assess the performance on clinical urine specimens.
We collected an additional urine specimen among individuals enrolling in an ongoing clinical trial at the Massachusetts General Hospital Sexual Health Clinic who presented with symptoms of urethritis or cervicitis (urethral or vaginal discharge, dysuria, or dyspareunia). We then assessed thermal, detergent, and combination DNA extraction conditions, varying the duration of heat at 95°C and concentration of Triton X. We assessed the efficacy of the various DNA extraction methods by quantitative polymerase chain reaction (qPCR). Once an extraction method was selected, we incubated samples for 90 minutes to permit isothermal recombinase polymerase amplification. We then assessed the performance of lateral flow Cas13a-based detection using our previously designed A probe and primer system for detection, comparing lateral flow results with NAAT results from clinical care.
We assessed DNA extraction conditions on 3 clinical urine specimens. There was no consistent significant difference in copies per microliter of DNA obtained using more or less heat. On average, we noted that 0.02% triton combined with 5 minutes of heating to 95°C resulted in the highest DNA yield, however, 0.02% triton alone resulted in a quantity of DNA that was above the previously determined analytic sensitivity of the assay. Given that detergent-based extraction is more easily deployable, we selected that as our method for extraction. We treated 23 clinical specimens with 0.02% triton, which we added to the Cas13a detection system. We ran all lateral flow detections in duplicate. The Cas13a-based assay detected 8 of 8 (100%) positive specimens, and 0 of 15 negative specimens.
Using point-of-care DNA extraction, isothermal amplification, and Cas13a-based detection, our point-of-care lateral flow assay correctly identified 23 clinical urine specimens as either positive or negative. Further evaluation of this assay among larger samples and more diverse sample types is warranted.
[疾病名称]的诊断依赖于核酸扩增检测（NAAT），而在感染率最高的资源有限环境中无法进行此类检测。利用CRISPR酶的高特异性在分子诊断方面的最新进展可以实现可现场部署的即时检测侧向流动分析。我们之前报道了一种用于检测[疾病名称]的侧向流动分析方法的开发和性能。在此，我们旨在将该分析方法与即时DNA提取技术相结合，并评估其在临床尿液样本上的性能。
我们在马萨诸塞州总医院性健康诊所正在进行的一项临床试验中，收集了出现尿道炎或宫颈炎症状（尿道或阴道分泌物、排尿困难或性交困难）的个体的额外尿液样本。然后，我们评估了热、去污剂和联合DNA提取条件，改变95°C加热的持续时间和Triton X的浓度。我们通过定量聚合酶链反应（qPCR）评估了各种DNA提取方法的有效性。一旦选择了一种提取方法，我们将样本孵育90分钟以进行等温重组酶聚合酶扩增。然后，我们使用之前设计的用于[疾病名称]检测的A探针和引物系统，评估基于Cas13a的侧向流动检测的性能，将侧向流动结果与临床护理中的NAAT结果进行比较。
我们在3份临床尿液样本上评估了DNA提取条件。使用或多或少加热获得的每微升DNA拷贝数没有一致的显著差异。平均而言，我们注意到0.02%的曲拉通与5分钟加热至95°C相结合可产生最高的DNA产量，然而，仅0.02%的曲拉通产生的DNA量高于该分析方法先前确定的分析灵敏度。鉴于基于去污剂的提取更易于部署，我们选择其作为提取方法。我们用0.02%的曲拉通处理了23份临床样本，并将其添加到Cas13a检测系统中。我们对所有侧向流动检测进行了重复操作。基于Cas13a的检测方法检测出8份阳性样本中的8份（100%），15份阴性样本中的0份。
通过即时DNA提取、等温扩增和基于Cas13a的检测，我们的即时侧向流动[疾病名称]检测方法正确地将23份临床尿液样本鉴定为阳性或阴性。有必要在更大样本和更多样化样本类型中对该检测方法进行进一步评估。
