Abstract:

Objective To develop a real?time PCR method for human Plasmodium spp. qantitative detection and species identificaton. Methods According to the sequence of Plasmodium 18S rRNA, the primer set was designed based on the genus?specific region around the species?specfic region. The PCR products were amplified and cloned into pGEM?T vector to produce standard plasmids of real?time PCR, and melting curve analysis was conducted following real?time PCR for Plamdium species indentification. Results By using the primer set, specific PCR products were produced from all of 4 human malaria parasites. The correlation of real?time PCR standard curve was good enough (r = -1.00) for quantitation. According to the melting curve analysis, the melting temperatures (Tm) of Plasmodium malariae, falciparum, ovale and vivax were significantly different, being 71.3, 72.8, 74.6 ℃ and 75.8 ℃, respectively. Conclusion The SYBR Green I based real?time PCR method developed in this study can be used for human Plasmodium spp. quantitative detection and species identificaton.

Key words: Plasmodium spp, Quantitative detection, Species identification, Real?time PCR

摘要：

目的 建立一种可用于疟原虫检测并同时鉴别虫种的荧光定量PCR方法。方法 根据人体疟原虫18S rRNA基因序列特征， 在疟原虫种特异性区域两侧的属特异性保守区设计引物， 对4种人体疟原虫进行PCR扩增， 将得到的扩增产物采用TA克隆法插入pGEM?T载体制备标准质粒， 用于制备标准曲线进行定量分析， 并进行熔解曲线分析， 测定各虫种扩增产物的熔解温度。结果 4种人体疟原虫均能扩增得到特异性片断， 荧光定量PCR方法中建立的标准曲线相关关系较好 （相关系数r = -1.00）， 熔解曲线分析结果显示， 4种人体疟原虫的熔解温度相差较大， 分别为三日疟原虫71.3 ℃、 恶性疟原虫72.8 ℃、 卵形疟原虫74.6 ℃和间日疟原虫75.8 ℃。 结论 建立的SYBR Green I染料法荧光定量PCR技术可同时进行人体疟原虫的定量检测和虫种鉴别。

关键词: 疟原虫；定量检测；虫种鉴别；荧光定量PCR