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Emergency SARS-CoV-2 Variants of Concern: Novel Multiplex Real-Time RT-PCR Assay for Rapid Detection and Surveillance.
Microbiol Spectr. 2022 02 23; 10(1):e0251321.MS

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide. Many variants of SARS-CoV-2 have been reported, some of which have increased transmissibility and/or reduced susceptibility to vaccines. There is an urgent need for variant phenotyping for epidemiological surveillance of circulating lineages. Whole-genome sequencing is the gold standard for identifying SARS-CoV-2 variants, which constitutes a major bottleneck in developing countries. Methodological simplification could increase epidemiological surveillance feasibility and efficiency. We designed a novel multiplex real-time reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2 variants with S gene mutations. This multiplex PCR typing method was established to detect 9 mutations with specific primers and probes (ΔHV 69/70, K417T, K417N, L452R, E484K, E484Q, N501Y, P681H, and P681R) against the receptor-binding domain of the spike protein of SARS-CoV-2 variants. In silico analyses showed high specificity of the assays. Variants of concern (VOC) typing results were found to be highly specific for our intended targets, with no cross-reactivity observed with other upper respiratory viruses. The PCR-based typing methods were further validated using whole-genome sequencing and a commercial kit that was applied to clinical samples of 250 COVID-19 patients from Taiwan. The screening of these samples allowed the identification of epidemic trends by time intervals, including B.1.617.2 in the third Taiwan wave outbreak. This PCR typing strategy allowed the detection of five major variants of concern and also provided an open-source PCR assay which could rapidly be deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, P.1, and B.1.617.2 variants and of four Omicron mutations on the spike protein (ΔHV 69/70, K417N, N501Y, P681H). IMPORTANCE COVID-19 has spread globally. SARS-CoV-2 variants of concern (VOCs) are leading the next waves of the COVID-19 pandemic. Previous studies have pointed out that these VOCs may have increased infectivity, have reduced vaccine susceptibility, change treatment regimens, and increase the difficulty of epidemic prevention policy. Understanding SARS-CoV-2 variants remains an issue of concern for all local government authorities and is critical for establishing and implementing effective public health measures. A novel SARS-CoV-2 variant identification method based on a multiplex real-time RT-PCR was developed in this study. Five SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, and Omicron) were identified simultaneously using this method. PCR typing can provide rapid testing results with lower cost and higher feasibility, which is well within the capacity for any diagnostic laboratory. Characterizing these variants and their mutations is important for tracking SAR-CoV-2 evolution and is conducive to public infection control and policy formulation strategies.

Authors+Show Affiliations

Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Division of Infectious Diseases and Tropical Medicine, Department of Medicine, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Division of Infectious Diseases and Tropical Medicine, Department of Medicine, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Division of Pulmonary and Critical Care Medicine, Department of Medicine, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Center for Precision Medicine and Genomics, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Division of Infectious Diseases and Tropical Medicine, Department of Medicine, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Department of Otolaryngology-Head and Neck Surgery, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospitalgrid.278244.f, National Defense Medical Center, Taipei, Taiwan, Republic of China.

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

35196812

Citation

Chung, Hsing-Yi, et al. "Emergency SARS-CoV-2 Variants of Concern: Novel Multiplex Real-Time RT-PCR Assay for Rapid Detection and Surveillance." Microbiology Spectrum, vol. 10, no. 1, 2022, pp. e0251321.
Chung HY, Jian MJ, Chang CK, et al. Emergency SARS-CoV-2 Variants of Concern: Novel Multiplex Real-Time RT-PCR Assay for Rapid Detection and Surveillance. Microbiol Spectr. 2022;10(1):e0251321.
Chung, H. Y., Jian, M. J., Chang, C. K., Lin, J. C., Yeh, K. M., Chen, C. W., Hsieh, S. S., Hung, K. S., Tang, S. H., Perng, C. L., Chang, F. Y., Wang, C. H., & Shang, H. S. (2022). Emergency SARS-CoV-2 Variants of Concern: Novel Multiplex Real-Time RT-PCR Assay for Rapid Detection and Surveillance. Microbiology Spectrum, 10(1), e0251321. https://doi.org/10.1128/spectrum.02513-21
Chung HY, et al. Emergency SARS-CoV-2 Variants of Concern: Novel Multiplex Real-Time RT-PCR Assay for Rapid Detection and Surveillance. Microbiol Spectr. 2022 02 23;10(1):e0251321. PubMed PMID: 35196812.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Emergency SARS-CoV-2 Variants of Concern: Novel Multiplex Real-Time RT-PCR Assay for Rapid Detection and Surveillance. AU - Chung,Hsing-Yi, AU - Jian,Ming-Jr, AU - Chang,Chih-Kai, AU - Lin,Jung-Chung, AU - Yeh,Kuo-Ming, AU - Chen,Chien-Wen, AU - Hsieh,Shan-Shan, AU - Hung,Kuo-Sheng, AU - Tang,Sheng-Hui, AU - Perng,Cherng-Lih, AU - Chang,Feng-Yee, AU - Wang,Chih-Hung, AU - Shang,Hung-Sheng, Y1 - 2022/02/23/ PY - 2022/2/24/entrez PY - 2022/2/25/pubmed PY - 2022/3/5/medline KW - E484K KW - E484R KW - K417N KW - K417T KW - L452R KW - N501Y KW - P681H KW - P681R KW - SARS-CoV-2 KW - VOC genotyping KW - epidemiological surveillance KW - ΔHV 69/70 SP - e0251321 EP - e0251321 JF - Microbiology spectrum JO - Microbiol Spectr VL - 10 IS - 1 N2 - Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide. Many variants of SARS-CoV-2 have been reported, some of which have increased transmissibility and/or reduced susceptibility to vaccines. There is an urgent need for variant phenotyping for epidemiological surveillance of circulating lineages. Whole-genome sequencing is the gold standard for identifying SARS-CoV-2 variants, which constitutes a major bottleneck in developing countries. Methodological simplification could increase epidemiological surveillance feasibility and efficiency. We designed a novel multiplex real-time reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2 variants with S gene mutations. This multiplex PCR typing method was established to detect 9 mutations with specific primers and probes (ΔHV 69/70, K417T, K417N, L452R, E484K, E484Q, N501Y, P681H, and P681R) against the receptor-binding domain of the spike protein of SARS-CoV-2 variants. In silico analyses showed high specificity of the assays. Variants of concern (VOC) typing results were found to be highly specific for our intended targets, with no cross-reactivity observed with other upper respiratory viruses. The PCR-based typing methods were further validated using whole-genome sequencing and a commercial kit that was applied to clinical samples of 250 COVID-19 patients from Taiwan. The screening of these samples allowed the identification of epidemic trends by time intervals, including B.1.617.2 in the third Taiwan wave outbreak. This PCR typing strategy allowed the detection of five major variants of concern and also provided an open-source PCR assay which could rapidly be deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, P.1, and B.1.617.2 variants and of four Omicron mutations on the spike protein (ΔHV 69/70, K417N, N501Y, P681H). IMPORTANCE COVID-19 has spread globally. SARS-CoV-2 variants of concern (VOCs) are leading the next waves of the COVID-19 pandemic. Previous studies have pointed out that these VOCs may have increased infectivity, have reduced vaccine susceptibility, change treatment regimens, and increase the difficulty of epidemic prevention policy. Understanding SARS-CoV-2 variants remains an issue of concern for all local government authorities and is critical for establishing and implementing effective public health measures. A novel SARS-CoV-2 variant identification method based on a multiplex real-time RT-PCR was developed in this study. Five SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, and Omicron) were identified simultaneously using this method. PCR typing can provide rapid testing results with lower cost and higher feasibility, which is well within the capacity for any diagnostic laboratory. Characterizing these variants and their mutations is important for tracking SAR-CoV-2 evolution and is conducive to public infection control and policy formulation strategies. SN - 2165-0497 UR - https://www.unboundmedicine.com/medline/citation/35196812/Emergency_SARS_CoV_2_Variants_of_Concern:_Novel_Multiplex_Real_Time_RT_PCR_Assay_for_Rapid_Detection_and_Surveillance_ DB - PRIME DP - Unbound Medicine ER -