客户采用我司硅羟基磁珠研究低载量新冠病毒检测技术在Analytica Chimica Acta发文

Importance of sample input volume for accurate SARS-CoV-2 qPCR testing
Yugan He，Tie Xie，Qihang Tu，Yigang Tong
https://doi.org/10.1016/j.aca.2022.339585

Abstract
Nucleic acid testing is the most widely used detection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Currently, a number of COVID-19 real-time quantitative reverse transcription PCR (qPCR) kits with high sensitivity and specificity are available for SARS-CoV-2 testing. However, these qPCR assays are not always reliable in detecting low viral load samples (Ct-value ≥ 35), resulting in inconclusive or false-negative results. Here, we used a Poisson distribution to illustrate the inconsistent performance of qPCR tests in detecting low viral load samples. From this, we concluded that the false-negative outcomes resulted from the random occurrences of sampling zero target molecules in a single test, and the probability to sample zero target molecules in one test decreased significantly with increasing purified RNA or initial sample input volume. At a given RNA concentration of 0.5 copy/μL, the probability of sampling zero RNA molecules decreased from 36.79% to close to 0.67% after increasing the RNA input volume from 2 to 10 μL. A SARS-CoV-2 qPCR assay with an LOD of 300 copies/mL was used to validate the improved consistency of the qPCR tests. We found that the false-negative qPCR results of clinical COVID-19 samples with a Ct ≥ 35 decreased by 50% after increasing the input of purified RNA from 2 to 10 μL. The consistency, accuracy, and robustness of nucleic acid testing for SARS-CoV-2 samples with low viral loads can be improved by increasing the sample input volume.

核酸检测是严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 最广泛使用的检测方法，它是 2019 年冠状病毒病 (COVID-19) 大流行的病原体。目前，许多具有高灵敏度和特异性的 COVID-19 实时定量逆转录 PCR (qPCR) 试剂盒可用于 SARS-CoV-2 检测。然而，这些 qPCR 检测在检测低病毒载量样本（Ct 值 ≥ 35）时并不总是可靠的，从而导致不确定或假阴性结果。在这里，我们使用泊松分布来说明 qPCR 测试在检测低病毒载量样本中的不一致性能。由此，我们得出结论，假阴性结果是由于在单次测试中随机发生零靶分子采样，并且在一次测试中采样零靶分子的概率随着纯化 RNA 或初始样本输入量的增加而显着降低。在 0.5 拷贝/μL 的给定 RNA 浓度下，在将 RNA 输入量从 2 增加到 10 μL 后，采样零 RNA 分子的概率从 36.79% 下降到接近 0.67%。 LOD 为 300 拷贝/mL 的 SARS-CoV-2 qPCR 测定用于验证 qPCR 测试的一致性改进。我们发现，在将纯化 RNA 的输入量从 2 μL 增加到 10 μL 后，Ct ≥ 35 的临床 COVID-19 样本的假阴性 qPCR 结果降低了 50%。通过增加样本输入量，可以提高病毒载量较低的 SARS-CoV-2 样本核酸检测的一致性、准确性和稳健性。

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