Epidemiology Unit, IRCCS Istituto Nazionale Tumori 'Fondazione G. Pascale', I‑80131 Naples, Italy.
Department of Biomedical and Biotechnological Sciences, Section of Microbiology, University of Catania, I‑95123 Catania, Italy.
Int J Mol Med. 2020 Sep;46(3):957-964. doi: 10.3892/ijmm.2020.4673. Epub 2020 Jul 13.
Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) is the gold standard method for the diagnosis of COVID‑19 infection. Due to pre‑analytical and technical limitations, samples with low viral load are often misdiagnosed as false‑negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT‑qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID‑19 were analyzed by droplet digital PCR (ddPCR) and RT‑qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)‑approved probe for the SARS‑CoV‑2 N gene were used. SYBR‑Green RT‑qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT‑qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT‑qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10‑fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT‑qPCR for the diagnosis of COVID‑19 infection in false‑negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID‑19 and the follow‑up of positive patients until complete remission.
逆转录-定量聚合酶链反应 (RT-qPCR) 是诊断 COVID-19 感染的金标准方法。由于分析前和技术限制，病毒载量低的样本常被误诊为假阴性样本。因此，评估其他能够克服 RT-qPCR 局限性的策略很重要。为了评估两种方法的灵敏度，对经临床诊断为 COVID-19 阳性和阴性的两个人的盲拭子样本进行了数字液滴 PCR (ddPCR) 和 RT-qPCR 分析。使用了嵌入化学法和世界卫生组织 (WHO)/疾病控制与预防中心 (CDC) 批准的 SARS-CoV-2 N 基因探针。SYBR-Green RT-qPCR 无法诊断病毒载量低的阳性样本，而 TaqMan 探针 RT-qPCR 在 Ct 值很晚时才给出阳性信号。相反，ddPCR 与 RT-qPCR 相比显示出更高的灵敏度，EvaGreen 和探针 ddPCR 均能够识别低病毒载量的样本为阳性，即使在 10 倍稀释浓度下也是如此。总之，ddPCR 显示出比 RT-qPCR 更高的灵敏度和特异性，可用于诊断病毒载量低的假阴性 COVID-19 感染样本。因此，ddPCR 强烈推荐用于 COVID-19 的诊断和阳性患者的随访，直至完全缓解。
