National Institute of Health-INS (Instituto Nacional de Salud-INS), Lima, Peru.
Pan American Health Organization-PAHO, Washington, DC, United States of America.
PLoS One. 2021 Mar 24;16(3):e0248885. doi: 10.1371/journal.pone.0248885. eCollection 2021.
One of the biggest challenges during the pandemic has been obtaining and maintaining critical material to conduct the increasing demand for molecular tests. Sometimes, the lack of suppliers and the global shortage of these reagents, a consequence of the high demand, make it difficult to detect and diagnose patients with suspected SARS-CoV-2 infection, negatively impacting the control of virus spread. Many alternatives have enabled the continuous processing of samples and have presented a decrease in time and cost. These measures thus allow broad testing of the population and should be ideal for controlling the disease. In this sense, we compared the SARS-CoV-2 molecular detection effectiveness by Real time RT-PCR using two different protocols for RNA extraction. The experiments were conducted in the National Institute of Health (INS) from Peru. We compared Ct values average (experimental triplicate) results from two different targets, a viral and internal control. All samples were extracted in parallel using a commercial kit and our alternative protocol-samples submitted to proteinase K treatment (3 μg/μL, 56°C for 10 minutes) followed by thermal shock (98°C for 5 minutes followed by 4°C for 2 minutes); the agreement between results was 100% in the samples tested. In addition, we compared the COVID-19 positivity between six epidemiological weeks: the initial two in that the Real time RT-PCR reactions were conducted using RNA extracted by commercial kit, followed by two other using RNA obtained by our kit-free method, and the last two using kit once again; they did not differ significantly. We concluded that our in-house method is an easy, fast, and cost-effective alternative method for extracting RNA and conducing molecular diagnosis of COVID-19.
在疫情期间，最大的挑战之一是获取和维持关键材料，以满足日益增长的分子检测需求。有时，由于高需求，供应商的缺乏和这些试剂的全球短缺使得难以检测和诊断疑似 SARS-CoV-2 感染的患者，从而对病毒传播的控制产生负面影响。许多替代方法使样本的连续处理成为可能，并降低了时间和成本。这些措施可以实现对人群的广泛检测，应该是控制疾病的理想选择。在这方面，我们比较了使用两种不同 RNA 提取方案的实时 RT-PCR 对 SARS-CoV-2 分子检测的有效性。实验在秘鲁国家卫生研究所 (INS) 进行。我们比较了两个不同靶标（病毒和内部对照）的实时 RT-PCR 实验的平均 Ct 值（实验重复三次）结果。所有样本均使用商业试剂盒和我们的替代方案平行提取 - 样本进行蛋白酶 K 处理（3μg/μL，56°C 10 分钟），然后进行热冲击（98°C 5 分钟，然后 4°C 2 分钟）；在测试的样本中，结果的一致性为 100%。此外，我们比较了六个流行病学周的 COVID-19 阳性率：最初的两周使用商业试剂盒提取的 RNA 进行实时 RT-PCR 反应，随后的两周使用我们的无试剂盒方法获得的 RNA 进行，最后两周再次使用试剂盒；它们没有显著差异。我们得出结论，我们的内部方法是一种简单、快速且具有成本效益的替代方法，可用于提取 RNA 并进行 COVID-19 的分子诊断。
