Accessible and Adaptable Multiplexed Real-Time PCR Approaches to Identify SARS-CoV-2 Variants of Concern

• DOI: 10.1128/spectrum.03222-22
• Publication Date: 2022-09-15T13:00:28Z
• Authors: Ting Yan; Ye Xu; Rongrong Zheng; Xiaohong Zeng; Zehui Chen; Su Lin; Zihan Xia; Yiqun Liao; Yongyou Zhang; Qingge Li
• Keywords: 0303 health sciences; rapid genotyping; SARS-CoV-2; SARS-CoV-2 VOCs; COVID-19; Omicron variant; multiplex PCR; Real-Time Polymerase Chain Reaction; Microbiology; QR1-502; 3. Good health; 03 medical and health sciences; Spike Glycoprotein, Coronavirus; Mutation; Humans; RNA, Viral; Pandemics; Research Article

Rapid identification and continuous surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are critical for guiding the response to COVID-19 pandemic. Whole-genome sequencing (WGS) is a preferred tool this aim, but many laboratories suffer from lack resources support population-level sequencing. Here, we describe two PCR strategies targeting spike protein mutations identify Alpha, Delta, Omicron variants. Signature were selected using dedicated bioinformatic program. The in Alpha Delta detected multicolor melting curve analysis (MMCA). Thirty-two variant targeted MeltArray approach one reaction, which was able detect subvariants BA.1, BA.2, BA.3, BA.4/5. limits detection varied five 50 copies RNA templates/reactions. No cross-reactivity observed with nine other viruses, including coronaviruses. We validated MMCA assays 309 SARS-CoV-2 positive samples collected at different time points. These exhibited 98.3% 100% sensitivity specificity compared WGS. Multiplexed real-time represent an alternative capable identifying current VOCs, adaptable emerging accessible existing equipment personnel. IMPORTANCE mutation VOCs crucial control, management, prevention. developed rapid molecular based on platform important concern, large number mutations. by R Then, established variants, assay 32 variant. multiplexed could be performed 96-well instrument within 2.5 h, offering high-throughput choice dynamic monitoring standard microbiology laboratory.