应用不依赖序列的单引物扩增技术获得人博卡病毒全基因序列

Sequence the complete sequence of bocavirus I with SISPA-PCR

目的 研究通过不依赖序列的单引物扩增（Sequence independent single primeramplification,SISPA）技术获得腹泻标本中人博卡病毒的全基因序列.方法 筛检仅含有人博卡病毒的标本,经过滤、超速离心富集和核酸酶处理后提取病毒核酸,采用SISPA-PCR技术扩增病毒序列,克隆后测序,经比对分析获得人博卡病毒基因序列,再用DNAstar拼接成全基因序列.结果 获得了人博卡病毒4834bp的序列,仅两端少部分序列未得到.结论 SISPA-PCR技术是一种可直接从样品中获得病毒全基因序列的方法.

Objective To sequence the complete sequence of bocavirus I with sequence independent single primer amplification （SISPA-PCR）. Methods To exclude the co-effection samples, all clinical samples of diarrhea cases were screened with special primers of rotavirus, astrovirus, adenovirus, calicivirus and bocavirus I . The virus were enriched through ultracentrifugation. Other nucleic acids,such as human and bacteria genomes, were degradated by DNase I and RNase. DNA of hocavirus was Amplificated with SISPA-PCR,then purificated ,cloned and sequenced. The sequences were alighmented in nr with blastn and assembled with DNAstar. Results A 4834bp sequence of hocavirus I were assembled. Conclusion SISPA-PCR is an economical and Efficient technique for sequence a virus complete genome.