苏丹型埃博拉病毒焦磷酸测序方法的建立

Establishment of Pyrosequencing Assay for Detection of Sudan Ebola Virus

[目的]本研究旨在通过对埃博拉病毒进行序列信息分析的基础上,利用焦磷酸测序技术对苏丹型埃博拉病毒进行快速检测和鉴定。[方法]通过序列信息比对,设计苏丹型埃博拉病毒基因保守区段的扩增引物及测序引物。通过人工方法合成一段基因序列,PCR扩增目的基因片段,经体外转录制备c RNA,RT-PCR扩增目的基因片段,采用焦磷酸测序技术针对目的基因进行保守核苷酸区段的测序分析。[结果]通过序列信息比对寻找到苏丹型埃博拉病毒基因型的核苷酸保守区段,经焦磷酸测序后能进一步确证毒株的序列信息为苏丹型埃博拉病毒。经过与华大基因公司进行Sanger法测序比较,结果完全一致。[结论]基于序列分析的焦磷酸测序技术可以作为进一步确证方法使用。

[Objective] To establish a new molecular method for simultaneous and rapid detection and confirmation of Ebola virus by using sequence analysis combined with pyrosequencing technology. [Methods] Amplification primers and a sequencing primer were designed according to the published sequences of conserved L gene regions of three Sudan Ebola viruses in GenBank. A gene sequence was synthesized artificially, and the target gene were amplified by PCR, to prepare cRNA through in vitro transcription. RT-PCR was developed for conserved regions of the gene, and the RT-PCR products were pysrosequenced.[Results] The characterized nucleotide segment was obtained by sequence analysis, and then the segment was confirmed as Sudan Ebola virus. Compared to the Sanger method of Huada, they showed 100% agreement.[Conclusion] Pyrosequencing technology based on sequence analysis can be used to confirm Sudan Ebola virus.