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

[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.

[学科分类] 兽医学

[关键词] amplification;genome;PRRSV;shotgun sequencing