恶性疟原虫荧光定量聚合酶链反应检测方法的建立

Fluorogenic quantitative PCR method for detecting plasmodium falciparum.

目的 建立恶性疟原虫荧光定量聚合酶链反应 (PCR)检测方法 ,为快速、准确地定量检测恶性疟原虫奠定基础。方法 根据恶性疟原虫SSUrRNA基因序列 ,设计并合成引物和荧光标记探针。将PCR扩增的恶性疟原虫SSUrRNA片段克隆入载体 ,重组质粒经筛选、鉴定后 ,作为阳性模板 ,用于标准曲线的制定和样品检测。结果 应用重组质粒制作的定量曲线循环阈值与模板浓度具有良好的线性关系 ,相关系数为 0 998。检测恶性疟血样 2 6份、间日疟血样 2 0份及混合感染血样 5份均阳性 ;正常血样 36份均阴性。结论 建立了检测恶性疟原虫的荧光定量PCR方法 ,较常规PCR技术更为简便、快速、准确 ,有很好的应用前景和研究价值。

Objective To establish fluorogenic quantitative PCR method for detecting Plasmodium falciparum.Method According to the specific sequence of Plasmodium falciparum SSUrRNA genes, the primers and the fluorogenic probe were designed and synthesized. The fragment generated from SSUrRNA as template was cloned into the T vector which was constructed from the pBluescript plasmid. The positive recombinant plasmid would be used as standard quantitative template to make the standard curve for sample detection.Results The standard curve indicated the linear relationship between CT(cycle threshold) and template concentration. The detection of 87 cases of P.f, Pv and normal blood samples proved the advantage of this method.Conclusion The fluorogenic Quantitative PCR method for the detection of Plasmodium falciparum is simple and accurate.