Virology : Concise and Broadly Applicable Method for Determining the Genomic Sequences of North-American–Type Porcine Reproductive and Respiratory Syndrome Viruses in Various Clusters

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  • MOROZUMI Takeya
    Animal Research Division, Institute of Japan Association for Techno-innovation in Agriculture, Forestry and Fisheries, 446–1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305–0854, Japan
  • ISEKI Hiroshi
    Viral Disease and Epidemiology Research Division, National Institute of Animal Health, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan
  • TOKI Daisuke
    Animal Research Division, Institute of Japan Association for Techno-innovation in Agriculture, Forestry and Fisheries, 446–1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305–0854, Japan
  • TAKAGI Michihiro
    Viral Disease and Epidemiology Research Division, National Institute of Animal Health, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan
  • TSUNEMITSU Hiroshi
    Viral Disease and Epidemiology Research Division, National Institute of Animal Health, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan Present address: Dairy Hygiene Research Division, National Institute of Animal Health, 4 Hitsujigaoka, Toyohira, Sapporo, Hokkaido 062–0045, Japan
  • UENISHI Hirohide
    Animal Immune and Cell Biology Research Unit, Division of Animal Sciences, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305–8602, Japan Animal Genome Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305–8602, Japan

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  • Concise and Broadly Applicable Method for Determining the Genomic Sequences of North-American–Type Porcine Reproductive and Respiratory Syndrome Viruses in Various Clusters

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We developed a concise and broadly applicable method for accurate genomic sequencing of North American genotype (NA-type) porcine reproductive and respiratory syndrome viruses (PRRSVs) that overcomes high genetic variability of the viruses. The method, designated “combination of consensus oligonucleotide reverse transcription and multiple displacement amplification” (CORT-MDA), involves reverse-transcription of viral RNA followed by shotgun sequencing after amplification using only 11 degenerate oligonucleotide primers; these primers were designed against consensus regions within the open reading frames of the 124 NA-type PRRSV strains with reported full-length genomic sequences. Sequencing of the 192 shotgun clones generated per virus showed 80% to 94% coverage on the reported PRRSV genomic sequence, such that only 2 or 3 unread regions had to be resequenced after PCR amplification using custom primers. Direct sequencing of RT-PCR products confirmed absolute consistency between sequences determined by the CORT-MDA method and those from RT-PCR. These results suggest that our method is applicable to diverse NA-type viruses.

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