Biometrology Group, Korea Research Institute of Standards and Science, Daejeon, Republic of Korea.
Biometrology Group, Korea Research Institute of Standards and Science, Daejeon, Republic of Korea; Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Republic of Korea.
J Virol Methods. 2024 May;326:114911. doi: 10.1016/j.jviromet.2024.114911. Epub 2024 Mar 4.
Quantitative PCR (qPCR) is the gold standard for detecting nucleic acid sequences specific to the target pathogen. For COVID-19 diagnosis, several molecular assays have been developed. In this study, we present an optimization strategy for the measurement of SARS-CoV-2 RNA via multiplex qPCR and digital PCR (dPCR). Compared to qPCR, both droplet and chip-based dPCR, which are known to be more sensitive and accurate, showed a better resilience to suboptimal assay compositions and cycling conditions following the proposed optimizations. In particular, the formation of heterodimers among assays greatly interfered with qPCR results, but only minimally with dPCR results. In dPCR, existing heterodimers lowered the PCR efficiency, producing a dampened fluorescent signal in positive partitions. This can be corrected by adjusting the PCR cycling conditions, after which dPCR shows the capability of measuring the expected copy number. In addition, we present a process to improve the existing RdRp assay by correcting the primer sequences and matching the melting temperature, ultimately producing highly sensitive and robust assays. The results of this study can reduce the cost and time of SARS-CoV-2 diagnosis while increasing accuracy. Furthermore, our results suggest that dPCR is a reliable method for the accurate measurement of nucleic acid targets.
实时荧光定量 PCR（qPCR）是检测目标病原体特定核酸序列的金标准。针对 COVID-19 诊断，已经开发了几种分子检测方法。在这项研究中，我们提出了一种通过多重 qPCR 和数字 PCR（dPCR）测量 SARS-CoV-2 RNA 的优化策略。与 qPCR 相比，基于液滴和芯片的 dPCR 由于其具有更高的灵敏度和准确性，因此在经过优化后，对非最佳检测成分和循环条件具有更好的适应性。特别是，检测之间形成的异二聚体极大地干扰了 qPCR 结果，但对 dPCR 结果的干扰极小。在 dPCR 中，现有的异二聚体降低了 PCR 效率，在阳性分区中产生了衰减的荧光信号。通过调整 PCR 循环条件可以纠正这种情况，之后 dPCR 显示出能够测量预期拷贝数的能力。此外，我们提出了一种通过校正引物序列并匹配熔解温度来改进现有 RdRp 检测方法的过程，最终产生高度敏感和稳健的检测方法。本研究的结果可以降低 SARS-CoV-2 诊断的成本和时间，同时提高准确性。此外，我们的结果表明，dPCR 是一种准确测量核酸靶标的可靠方法。
