Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay
Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay
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DOI：
10.1016/j.jviromet.2020.113926
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发表时间：
2020-10-01
影响因子：
3.1
通讯作者：
Omata, Masao
中科院分区：
文献类型：
--
作者：
Hirotsu, Yosuke;Mochizuki, Hitoshi;Omata, Masao
背景：严重急性呼吸综合征冠状病毒2（SARS-CoV-2）在中国湖北省武汉市出现，已在全球传播并威胁人类生命。 SARS-CoV-2 的检测对于预防新的疫情爆发、遏制疾病传播和管理患者至关重要。目前，临床实验室使用逆转录聚合酶链反应（RT-PCR）检测来检测病毒。然而，尽管这种检测被认为具有高特异性，但据报道其灵敏度低至 60-70%。因此，迫切需要提高灵敏度。方法：我们使用美国CDC（N1、N2和N3）和日本NIID（N1和N2）推荐的引物和单猝灭探针。此外，我们根据 NIID 提供的病毒序列设计了双猝灭剂探针，以开发进一步的检测（称为 YCH 检测 [N1 和 N2]）。使用这些检测方法，我们使用连续稀释的 DNA 阳性对照进行 RT-PCR，以评估和比较三种检测方法的检测灵敏度。此外，还测试了 66 个鼻咽拭子以确定诊断性能。结果：与 NIID 检测相比，CDC 和 YCH 检测的 RT-PCR 阈值循环 (Ct) 值相对较低。系列稀释测定表明，CDC 和 YCH 测定均可检测低拷贝数的 DNA 阳性对照。与 NIID 测定相比，YCH 测定的基线背景荧光信号较低。我们使用 66 个鼻咽拭子评估了单 (NIID) 和双猝灭剂 (YCH) 探针之间的诊断性能。当使用 YCH-N2 检测结果作为参考时，每次检测均检测到 SARS-CoV-2，NIID-N1、YCH-N1、NIID-N2 的阳性百分比一致率为 56%、YCH-N1 为 61%、NIID-N2 为 94%，NIID-N1、YCH-N1 和 NIID-N2 的阴性百分比一致率为 100%。结论：双猝灭剂探针降低了背景荧光并改善了荧光强度。 RT-PCR 对 SARS-CoV-2 的检测敏感性。
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in the city of Wuhan, Hubei Province, China, has spread worldwide and is threatening human life. The detection of SARS-CoV-2 is critical for preventing new outbreaks, curbing disease spread, and managing patients. Currently, a reverse-transcription polymerase chain reaction (RT-PCR) assay is used to detect the virus in clinical laboratories. However, although this assay is considered to have high specificity, its sensitivity is reportedly as low as 60-70 %. Improved sensitivity is, therefore, urgently required.Methods: We used the primers and single-quencher probes recommended by the CDC (N1, N2 and N3) in the USA and the NIID (N1 and N2) in Japan. In addition, we designed double-quencher probes according to the virus sequence provided by the NIID to develop a further assay (termed the YCH assay [N1 and N2]). Using these assays, we conducted RT-PCR with serially diluted DNA positive controls to assess and compare the detection sensitivity of the three assays. Furthermore, 66 nasopharyngeal swabs were tested to determine the diagnostic performances.Results: The threshold cycle (Ct) value of the RT-PCR was relatively low for the CDC and YCH assays compared with the NIID assay. Serial dilution assays showed that both the CDC and YCH assays could detect low copy numbers of the DNA positive control. The background fluorescence signal at the baseline was lower for the YCH assay compared with the NIID assay. We assessed the diagnostic performance between single- (NIID) and double-quencher (YCH) probes using 66 nasopharyngeal swabs. When the results of YCH-N2 assay were used as a reference, each assay detected SARS-CoV-2 with positive percent agreements of 56 % for NIID-N1, 61 % for YCH-N1, and 94 % for NIID-N2, and 100 % negative percent agreements for NIID-N1, YCH-N1 and NIID-N2.Conclusion: Double-quencher probes decreased the background fluorescence and improved the detection sensitivity of RT-PCR for SARS-CoV-2.