期刊
JOURNAL OF VETERINARY MEDICAL SCIENCE
卷 76, 期 9, 页码 1249-1255出版社
JAPAN SOC VET SCI
DOI: 10.1292/jvms.14-0103
关键词
amplification; genome; PRRSV; shotgun sequencing
资金
- Ministry of Agriculture, Forestry and Fisheries of Japan [AGB1003]
- Japan Racing Association
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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