A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection.
A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection.
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DOI：
10.1371/journal.pbio.3001030
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发表时间：
2020-12
期刊：
影响因子：
9.8
通讯作者：
Jackson AP
中科院分区：
文献类型：
--
作者：
Reijns MAM;Thompson L;Acosta JC;Black HA;Sanchez-Luque FJ;Diamond A;Parry DA;Daniels A;O'Shea M;Uggenti C;Sanchez MC;O'Callaghan A;McNab MLL;Adamowicz M;Friman ET;Hurd T;Jarman EJ;Chee FLM;Rainger JK;Walker M;Drake C;Longman D;Mordstein C;Warlow SJ;McKay S;Slater L;Ansari M;Tomlinson IPM;Moore D;Wilkinson N;Shepherd J;Templeton K;Johannessen I;Tait-Burkard C;Haas JG;Gilbert N;Adams IR;Jackson AP
随着由新型冠状病毒 SARS-CoV-2（严重急性呼吸系统综合症冠状病毒 2）引起的 COVID-19（2019 冠状病毒病）大流行，需要敏感、特异且经济实惠的诊断测试来识别感染者（并非所有感染者都有症状）。最灵敏的测试涉及使用 RT-qPCR（定量逆转录 PCR）检测病毒 RNA，现在有许多商业试剂盒可用于此目的。然而，这些试剂盒价格昂贵，并且不能总是保证提供足够数量的此类试剂盒。因此，我们开发了一种多重检测方法，使用成熟的 SARS-CoV-2 靶标以及人类细胞对照 (RPP30) 和病毒掺入对照（Phocine Herpes Virus 1 [PhHV-1]），分别监测样品质量和核酸提取效率。在这里，我们确定该测试的性能与广泛使用的商业检测一样，但成本大大降低。此外，我们证明通过鼻子和喉咙联合拭子常规收集的材料存在 >1,000 倍的变异性，并在检测到的人类核酸和 SARS-CoV-2 核酸水平之间建立了统计学上显着的相关性。因此，在我们的检测中加入人类对照探针提供了样品质量的定量测量，有助于降低假阴性率。我们证明了建立强大的 RT-qPCR 检测方法的可行性，其成本约为同等商业检测方法的 10%，这可以使资源匮乏的环境受益，并使大批量检测变得经济实惠。需要更好、更便宜的 SARS-CoV-2 qRT-PCR 检测，但众所周知，拭子样本中的人类和病毒核酸量是相关的，这显示了人类质量控制探针的重要性。这项研究描述了多重检测，其性能与商业测试相同，但成本约为商业测试的 10%。
With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable. Better and cheaper SARS-CoV-2 qRT-PCR tests are needed, but it is known that human and viral nucleic acid quantities in swab samples correlate, showing the importance of a human quality control probe. This study describes multiplex assays that perform equally well to commercial tests, but at ~10% of the cost.
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发表时间：
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期刊：
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影响因子：
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