Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification
Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification
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DOI：
10.1093/clinchem/hvaa267
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发表时间：
2021-02-01
影响因子：
9.3
通讯作者：
Milbrandt, Jeffrey
中科院分区：
文献类型：
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作者：
Lalli, Matthew A.;Langmade, S. Joshua;Milbrandt, Jeffrey
背景：需要快速、可靠和广泛的检测来遏制持续的COVID-19大流行。目前的金标准核酸检测受到供应短缺的关键试剂，包括鼻拭子，RNA提取试剂盒，个人防护设备，仪器，和labor.METHODS：为了克服这些挑战，我们开发了一种快速比色测定使用逆转录环介导等温扩增（RT-LAMP）优化的人唾液样本没有RNA纯化步骤。我们描述了唾液预处理方案的优化，以使RT-LAMP分析敏感的病毒检测。我们优化了RT-LAMP反应条件，并实施了高通量无偏方法进行检测解释。我们测试了唾液预处理是否也可以通过常规逆转录定量聚合酶链反应（RT-qPCR）进行病毒检测。最后，我们验证了这些检测对临床样品。结果：优化的唾液预处理方案，使分析灵敏的检测SARS-CoV-2从唾液中的比色RT-LAMP或RT-qPCR的提取。在模拟样品中，优化的RT-LAMP测定具有59（95%置信区间：44-104）个粒子拷贝/反应的检测限。我们强调了使用3个读数的LAMP测定实施的灵活性：裸眼比色法、分光光度法和实时荧光。在一组30份临床唾液样本中，直接对预处理的唾液样本进行比色RT-LAMP和RT-qPCR检测，无需提取RNA，准确率大于90%.CONCLUSIONS：比色RT-LAMP快速检测唾液中的SARS-CoV-2是一种简单、灵敏、经济有效的方法，具有广泛的潜力，可扩大对引起COVID-19的病毒的诊断测试。
BACKGROUND: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor.METHODS: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples.RESULTS: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%.CONCLUSIONS: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.