Srivatsan Sanjay, Heidl Sarah, Pfau Brian, Martin Beth K, Han Peter D, Zhong Weizhi, van Raay Katrina, McDermot Evan, Opsahl Jordan, Gamboa Luis, Smith Nahum, Truong Melissa, Cho Shari, Barrow Kaitlyn A, Rich Lucille M, Stone Jeremy, Wolf Caitlin R, McCulloch Denise J, Kim Ashley E, Brandstetter Elisabeth, Sohlberg Sarah L, Ilcisin Misja, Geyer Rachel E, Chen Wei, Gehring Jase, Kosuri Sriram, Bedford Trevor, Rieder Mark J, Nickerson Deborah A, Chu Helen Y, Konnick Eric Q, Debley Jason S, Shendure Jay, Lockwood Christina M, Starita Lea M
Department of Genome Sciences, University of Washington, Seattle, WA.
Brotman Baty Institute for Precision Medicine, Seattle, WA.
Clin Chem. 2021 Dec 30;68(1):143-152. doi: 10.1093/clinchem/hvab132.
The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse-transcription PCR (RT-qPCR). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction.
We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance.
After optimization, SwabExpress has a low limit of detection at 2-4 molecules/µL, 100% sensitivity, and 99.4% specificity when compared side by side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time.
SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.
由于迫切需要大规模开展针对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的临床检测，同时全球关键试剂和耗材短缺，因此有必要开发简化的实验室检测方案。传统的SARS-CoV-2核酸检测包括用鼻咽拭子在转运培养基中采集临床标本、核酸提取以及定量逆转录聚合酶链反应（RT-qPCR）。随着检测在全球范围内的扩大，全球供应链不堪重负，导致检测试剂和材料短缺。为解决短缺问题，我们开发了SwabExpress，这是一种端到端的方案，旨在使用大量生产的前鼻孔拭子，无需转运培养基和核酸提取。
我们评估了干燥运输并在低TE缓冲液中洗脱的前鼻孔拭子，将其作为依赖提取的病毒转运培养基的直接RT-qPCR替代方法。我们验证了使用热处理进行病毒灭活的方案，并增加了蛋白酶K消化步骤以减少扩增干扰。我们在存档和前瞻性收集的拭子标本上测试了该方案，以微调检测性能。
优化后，与采用提取的传统RT-qPCR方案相比，SwabExpress的检测下限低至2-4个分子/微升，灵敏度为100%，特异性为99.4%。在实际标本上，SwabExpress的性能优于自动化提取系统，同时降低了成本和操作时间。
SwabExpress是一种简化的工作流程，可在不牺牲检测性能的情况下促进新型冠状病毒肺炎（COVID-19）的大规模检测。它可作为简化基于PCR的临床实验室检测的模板，尤其是在大流行期间关键短缺时期。
