Double-Quencher Probes Improved the Detection Sensitivity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by One-Step RT-PCR

ABSTRACT

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerges in Wuhan City, Hubei Province, spreads worldwide, and threats the human life. The detection of SARS-CoV-2 is important for the prevention of the outbreak and management of patients. Real-time reverse-transcription polymerase chain reaction (RT-PCR) assay detected the virus in clinical laboratory. MethodsThis study utilized primers and single-quencher probes in accordance with the Centers for Disease Control and Prevention (CDC) in the USA and the National Institute of Infectious Diseases (NIID) in Japan. Moreover, we designed the double-quencher probes (YCH assay) according to the oligonucleotide sequence established by NIID. Using these assays, we conducted a one-step real-time RT-PCR with serial DNA positive control to assess the detection sensitivity. ResultsThe threshold cycle (Ct) value of RT-PCR was relatively low in CDC and YCH assays compared to NIID assay. Serial dilution assay showed that both CDC and YCH assays could detect a low-copy number of DNA positive control. The background fluorescent signal at the baseline was lower in YCH than that of NIID. ConclusionDouble-quencher probes decreased background fluorescent signal and improved detection sensitivity of SARS-CoV-2.