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

Yosuke Hirotsu; Hitoshi Mochizuki; Masao Omata

We also originally designed double-quencher probes (IDT) containing the same sequence in accordance with the NIID protocol (hereafter called YCH assay). This probe has 5' FAM dye, internal ZEN Quencher, and 3' Iowa Black® Fluorescent Quencher (IBFQ) ( Table 1) . Internal ZEN Quencher is incorporated between the ninth and tenth bases from the 5' end of probe. This design decreased the distance between the dye and quencher and expected to reduce the background signal and achieve a higher dynamic signal. All primer/probe sets of CDC, NIID, and YCH assays amplified the nucleocapsid (N) gene of SARS-CoV-2 ( Figure 1 ). CDC assay targets three sites of N gene, while both NIID and YCH target the two sites ( Figure 1 ).