Detection of West Nile Virus Using SYBR GreenⅠ Fluorescent Quantitative PCR Assay / 中华医院感染学杂志

ABSTRACT

OBJECTIVE To establish SYBR Green Ⅰ fluorescent quantitative PCR assay for the detection of West Nile virus(WNV),which could be used for early laboratory diagnosis.METHODS A fragment of WNV gene was amplified by PCR,then cloned into pMD-18 T vector.The combinant plasmid was sequenced and analyzed by means of BLAST program,and used as the positive DNA in place of WNV.The SYBR GreenⅠfluorescent quantitative PCR assay was established based on positive plasmid.The sensitivity and specificity of the assay were performed.RESULTS The combinant plasmid was confirmed by sequencing and the fragment belonged to WNV.Ten copies of WNV RNA were detected by SYBR GreenⅠfluorescent quantitative RT-PCR assay.Results of the other members of Flaviviridae were negative,which indicated this assay was specific for WNV.CONCLUSIONS SYBR GreenⅠfluorescent quantitative PCR assay established in this study is highly sensitive and specific,and so it can be used for early diagnosis of WNV infection.