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微芯片 RT-PCR 检测鼻咽 SARS-CoV-2 样本。

Microchip RT-PCR Detection of Nasopharyngeal SARS-CoV-2 Samples.

机构信息

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia.

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia.

出版信息

J Mol Diagn. 2021 Jun;23(6):683-690. doi: 10.1016/j.jmoldx.2021.02.009. Epub 2021 Mar 9.

Abstract

Fast, accurate, and reliable diagnostic tests are critical for controlling the spread of the coronavirus disease 2019 (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The current gold standard for testing is real-time PCR; however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 minutes), user-friendly, and accurate microchip real-time PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips preloaded with COVID-19 primers and probes for the N gene accommodate 1.2-μL reaction volumes, lowering the required reagents by 10-fold compared with tube-based real-time PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip real-time PCR provides a significantly lower resource alternative to the Centers for Disease Control and Prevention-approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.

摘要

快速、准确、可靠的诊断检测对于控制 2019 年冠状病毒病(COVID-19)的传播至关重要,COVID-19 与严重急性呼吸综合征冠状病毒 2(SARS-CoV-2)感染有关。目前用于检测的金标准是实时聚合酶链反应(PCR);然而,在当前的大流行期间,检测试剂盒和试剂的供应一直受到限制。我们报告了一种快速(30 分钟)、用户友好且准确的微芯片实时 PCR 检测方法,用于从鼻咽拭子 RNA 提取物中检测 SARS-CoV-2。微芯片预先加载了用于 N 基因的 COVID-19 引物和探针,可容纳 1.2 μL 的反应体积,与基于管的实时 PCR 相比,所需试剂减少了 10 倍。我们使用模拟参考样本和来自加拿大患者的 21 个临床样本验证了我们的检测方法,确定了每个反应 1 个拷贝的检测限。微芯片实时 PCR 为疾病预防控制中心批准的实时 RT-PCR 检测提供了一种明显更低的资源替代方案,具有可比的灵敏度,对临床样本的阳性和阴性预测一致性为 100%。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/cb63/7939975/3a6150600d24/gr1_lrg.jpg

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