SwabExpress: An End-to-End Protocol for Extraction-Free COVID-19 Testing
SwabExpress: An End-to-End Protocol for Extraction-Free COVID-19 Testing
复制标题
DOI：
10.1093/clinchem/hvab132
复制
发表时间：
2021-08-12
影响因子：
9.3
通讯作者：
Starita, Lea M.
中科院分区：
文献类型：
--
作者：
Srivatsan, Sanjay;Heidl, Sarah;Starita, Lea M.
背景：对 SARS-CoV-2 进行大规模临床检测的迫切需要，以及全球关键试剂和用品的短缺，需要制定简化的实验室检测方案。 SARS-CoV-2 的常规核酸检测包括使用鼻咽拭子在运输介质中采集临床标本、核酸提取和定量逆转录 PCR (RT-qPCR)。随着检测规模在全球范围内扩大，全球供应链已经崩溃，导致检测试剂和材料稀缺。为了解决短缺问题，我们开发了 SwabExpress，这是一种端到端方案，旨在采用大规模生产的前鼻孔拭子，并绕过传输介质和核酸提取的要求。方法：我们评估了前鼻孔拭子，干燥运输并在低 TE 缓冲液中洗脱，作为提取依赖性病毒传输介质的直接 RT-qPCR 替代品。我们验证了使用热处理进行病毒灭活的方案，并添加了蛋白酶 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样本中测试了该方案，以微调测试性能。结果：优化后，与采用提取的传统 RT-qPCR 方案相比，SwabExpress 的检测下限为 2-4 个分子/mL，灵敏度为 100%，特异性为 99.4%。在真实标本上，SwabExpress 的性能优于自动提取系统，同时降低了成本和操作时间。结论：SwabExpress 是一种简化的工作流程，可在不牺牲测试性能的情况下促进 COVID-19 的大规模测试。它可以作为简化基于 PCR 的临床实验室测试的模板，特别是在大流行期间严重短缺的时期。
BACKGROUND: 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.METHODS: 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.RESULTS: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/mL, 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.CONCLUSION: 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.