Статья

Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses

K. Zlateva, K. Crusio, A. Leontovich, C. Lauber, E. Claas, A. Kravchenko, W. Spaan, A. Gorbalenya,
2021

The ssRNA+ family Coronaviridae includes two subfamilies prototyped by coronaviruses and toroviruses that cause respiratory and enteric infections. To facilitate the identification of new distantly related members of the family Coronaviridae, we have developed a molecular assay with broad specificity. The consensus-degenerated hybrid oligonucleotide primer (CODEHOP) strategy was modified to design primers targeting the most conserved motifs in the RNA-dependent RNA polymerase locus. They were evaluated initially on RNA templates from virus-infected cells using a two-step RT-PCR protocol that was further advanced to a one-step assay. The sensitivity of the assay ranged from 10 2 to 10 6 and from 10 5 to 10 9 RNA copy numbers for individual corona-/torovirus templates when tested, respectively, with and without an excess of RNA from human cells. This primer set compared to that designed according to the original CODEHOP rules showed 10-10 3 folds greater sensitivity for 5 of the 6 evaluated corona-/torovirus templates. It detected 57% (32 of 56) of the respiratory specimens positive for 4 human coronaviruses, as well as stool specimens positive for a bovine torovirus. The high sensitivity and broad virus range of this assay makes it suitable for screening biological specimens in search for new viruses of the family Coronaviridae. © 2011 Elsevier B.V.

Цитирование

Похожие публикации

Источник

Версии

  • 1. Version of Record от 2021-04-27

Метаданные

Об авторах
  • K. Zlateva
    Department of Medical Microbiology, Leiden University Medical Center, 2300 RC Leiden, Netherlands
  • K. Crusio
    The A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119899 Moscow, Russian Federation
  • A. Leontovich
    Faculty of Bioengineering and Bioinformatics, M.V. Lomonosov Moscow State University, 119899 Moscow, Russian Federation
  • C. Lauber
  • E. Claas
  • A. Kravchenko
  • W. Spaan
  • A. Gorbalenya
Название журнала
  • Journal of Virological Methods
Том
  • 177
Выпуск
  • 2
Страницы
  • 174-183
Ключевые слова
  • primer RNA; RNA directed RNA polymerase; analytic method; animal cell; article; consensus degenerated hybrid oligonucleotide primer technique; controlled study; Coronavirus; cow; feces; gene amplification; gene cluster; gene locus; genetic distance; human; human cell; nonhuman; nucleotide sequence; phylogeny; priority journal; sensitivity and specificity; strain difference; Togavirus; validation study; virus detection; virus strain; Amino Acid Sequence; Animals; Cattle; Cell Line; Conserved Sequence; Coronavirus; Coronavirus Infections; DNA Primers; Humans; Molecular Sequence Data; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity; Sequence Alignment; Torovirus; Torovirus Infections; Virus Cultivation; Bovine torovirus; Coronaviridae; Coronavirus; Torovirus
Тип документа
  • journal article
Источник
  • scopus