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Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima.
Ko, Ko; Takahashi, Kazuaki; Nagashima, Shintaro; E, Bunthen; Ouoba, Serge; Takafuta, Toshiro; Fujii, Yoshiki; Mimori, Michi; Okada, Fumie; Kishita, Eisaku; Ariyoshi, Kunie; Hussain, Md Razeen Ashraf; Sugiyama, Aya; Akita, Tomoyuki; Kuwabara, Masao; Tanaka, Junko.
  • Ko K; Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
  • Takahashi K; Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
  • Nagashima S; Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
  • E B; Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
  • Ouoba S; Payment Certification Agency, Ministry of Health, Lot 80, Street 289, Sangkat Boeng Kak Ti Pir, Khan Tuol Kouk, Phnom Penh 12152, Cambodia.
  • Takafuta T; Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
  • Fujii Y; Unité de Recherche Clinique de Nanoro (URCN), Nanoro BP18, Burkina Faso.
  • Mimori M; Hiroshima City Funairi Citizens Hospital, 14-11, Funairisaiwaicho, Naka-ku, Hiroshima 730-0844, Japan.
  • Okada F; Hiroshima City Institute of Public Health, 4 Chome-1-2, Shoko Center, Nishi-ku, Hiroshima 733-0833, Japan.
  • Kishita E; Hiroshima City Health and Welfare Bureau, 1 Chome-6-34, Kokutaijimachi, Naka-ku, Hiroshima 734-0042, Japan.
  • Ariyoshi K; Hiroshima Prefectural Health and Welfare Bureau, 10-52, Motomachi, Naka-ku, Hiroshima 730-8511, Japan.
  • Hussain MRA; Hiroshima Prefectural Health and Welfare Bureau, 10-52, Motomachi, Naka-ku, Hiroshima 730-8511, Japan.
  • Sugiyama A; Hiroshima Prefectural Technology Research Institute, Public Health and Environment Center, 1 Chome-6-29, Minamimachi, Minami-ku, Hiroshima 734-0007, Japan.
  • Akita T; Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
  • Kuwabara M; Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
  • Tanaka J; Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Viruses ; 14(4)2022 03 30.
Article in English | MEDLINE | ID: covidwho-1820406
ABSTRACT
This study aimed to exercise the Sanger sequencing strategy for screening of variants among confirmed COVID-19 cases and validate our strategy against NGS strains in Hiroshima retrieved from GISAID. A total of 660 samples from confirmed COVID-19 cases underwent screening for variants by Sanger-based partial sequencing to the targeted spike gene (nt22,735~nt23,532) using an in-house-developed primer set. The identification of variants was done by unique checkpoints of base nucleotide changes in the targeted spike gene. Moreover, we amplified one full-length genome using Sanger method and an in-house-developed primer library. Using NGS strains of the same sampling period from GISAID, a phylogenetic tree was constructed to examine the distribution pattern of variants in Hiroshima and to validate our Sanger method. The modified primer set provided 100% validation and 99.2% amplification. PANGO Lineage R.1 was detected in late in the third wave, followed by Alpha (B.1.1.7) domination in the fourth wave, Delta (B.1.617.2) domination in the fifth wave, and Omicron (B.1.1.529) domination in the sixth wave, and there was no significant difference in viral copies between variants (p = 0.09). The variants showed different transmission patterns, but the distribution of variants is consistent to that shown by the phylogenetic tree. The Sanger method also provided successful amplification of the full-length genome of the SARS-CoV-2 virus. Our Sanger sequencing strategy was useful for the screening of SASR-CoV-2 variants without the need for full-genome amplification. The modified primer set was validated to use universally, which allows an understanding of the variants' distribution in real time and provides the evidence for policy-making and the formulation or modification of preventive strategies.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Observational study / Prognostic study / Randomized controlled trials Topics: Variants Limits: Humans Language: English Year: 2022 Document Type: Article Affiliation country: V14040720

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Observational study / Prognostic study / Randomized controlled trials Topics: Variants Limits: Humans Language: English Year: 2022 Document Type: Article Affiliation country: V14040720