Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, 92697, USA.
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA, 92697, USA.
Sci Rep. 2022 Mar 10;12(1):3951. doi: 10.1038/s41598-022-07871-4.
The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.
SARS-CoV-2 大流行凸显了快速诊断检测的必要性。大规模检测能力的成本和可用性导致周转时间滞后，阻碍了接触者追踪工作，从而进一步导致了 SARS-CoV-2 的传播。为了提高检测速度和频率，我们开发了一种经济高效的单管方法，用于采集、变性和分析临床样本。该方法使用 1 µL 微生物接种环采集唾液，使用十二烷基硫酸钠 (SDS) 使病毒基因组 RNA 失活并释放，然后使用含有聚山梨酯 80 (吐温 80) 的诊断反应混合物。在同一个管中，将 SDS 变性的临床样本引入含有所有核酸检测成分和吐温 80 胶束的混合物中，以吸收 SDS 并允许酶反应进行，从而无需进一步处理样本。测试人员可以自行采集样本，进一步减少了对训练有素的人员进行测试的需求。我们使用逆转录定量实时聚合酶链反应 (RT-qPCR) 和逆转录环介导等温扩增 (RT-LAMP) 验证了这种单管样本检测方法，发现与对照反应相比，含有吐温 80 和 SDS 的反应混合物之间几乎没有差异。这种方法减轻了传统大规模检测的后勤负担，并提供了一种可部署的即时护理诊断方法，以提高检测频率。
