Concise and Broadly Applicable Method for Determining the Genomic Sequencesof North-American–Type Porcine Reproductive and Respiratory Syndrome Viruses in VariousClusters

Journal of Veterinary Medical Science

Takeya MOROZUMI³  Michihiro TAKAGI²  Hiroshi ISEKI²  Hiroshi TSUNEMITSU²  Daisuke TOKI³  Hirohide UENISHI¹ 

[1] Animal Immune and Cell Biology Research Unit, Division of Animal Sciences, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305�?8602, Japan;Viral Disease and Epidemiology Research Division, National Institute of Animal Health, 3�?1�?5 Kannondai, Tsukuba, Ibaraki 305�?0856, Japan;Animal Research Division, Institute of Japan Association for Techno-innovation in Agriculture, Forestry and Fisheries, 446�?1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305�?0854, Japan

关键词: amplification;    genome;    PRRSV;    shotgun sequencing;   

DOI  :  10.1292/jvms.14-0103

学科分类：兽医学

来源: Japanese Society of Veterinary Science

【 摘 要 】

References(31)Supplementary materials(2)We developed a concise andbroadly applicable method for accurate genomic sequencing of North American genotype(NA-type) porcine reproductive and respiratory syndrome viruses (PRRSVs) that overcomeshigh genetic variability of the viruses. The method, designated “combination of consensusoligonucleotide reverse transcription and multiple displacement amplification�? (CORT-MDA),involves reverse-transcription of viral RNA followed by shotgun sequencing afteramplification using only 11 degenerate oligonucleotide primers; these primers weredesigned against consensus regions within the open reading frames of the 124 NA-type PRRSVstrains with reported full-length genomic sequences. Sequencing of the 192 shotgun clonesgenerated 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 usingcustom primers. Direct sequencing of RT-PCR products confirmed absolute consistencybetween sequences determined by the CORT-MDA method and those from RT-PCR. These resultssuggest that our method is applicable to diverse NA-type viruses.

【 授权许可】

Unknown