Abstract:

Objective To establish a fluorescent quantitative PCR（FQ?PCR）method for quantitative detection and species identification of Plasmodium sporozoites in Anopheles mosquitoes. Methods One pair of human Plasmodium genus?specific prim? ers based on 18S rRNA genes were used and the reaction system and reaction condition of FQ?PCR were optimized by using the mixture of Plasmodium 18S rRNA gene recombinant plasmids and Anopheles DNA as a template. The specificity was verified by us? ing four Plasmodium spp. 18S rRNA gene plasmid DNA as well as mosquito DNA and the Plasmodium species was identified ac? cording to the value of melting temperature（Tm） . The standard curve was made by using P. vivax 18S rRNA gene recombinant plasmids which were serially diluted by negative Anopheles DNA as a template. The sensitivity was analysed by using plasmid DNA and laboratory infected sporozoite positive mosquito DNA，respectively. The different parts and different amounts of Anopheles DNA were added into the reaction system to investigate the influence of Anopheles DNA on the assessment. Results There was no specific amplification for mosquito DNA and human blood DNA. There was specific amplification for Plasmodium 18S RNA gene recombinant plasmids and the Tm （s）of P. malariae，P. falciparum，P. ovale and P. vivax were 71.0，72.7，73.9 ℃ and 75.9℃，respectively，which were easy to be identified. The standard curve indicated a good linear relationship between the cycle threshold （Ct）and template concentration（r = -0.99） . The sensitivity was 50 copies of plasmid DNA or one sporozoite positive mosquito DNA diluted by 32 times of mosquito DNA. Anopheles DNA could inhibite the FQ?PCR reaction. The Ct value of amplification showed a good reproducibility both within the same experiment and among different experiments. Conclusion The novel SYBR Green I based FQ?PCR method developed in this study shows a high sensitivity and specificity and it can be used for quantitative detection and species identification of sporozoites in mosquitoes.

Key words: Fluorescent quantitative PCR（FQ?PCR）；Anopheles mosquito； Plasmodium sporozoite；Species identification

摘要：

目的 建立一种用于按蚊体内疟原虫子孢子定量检测和虫种鉴别的荧光定量PCR方法。方法 采用针对4种人体疟原虫18S rRNA基因属特异性保守区的1对引物， 以疟原虫18S rRNA基因重组质粒与按蚊DNA的混合物为模板， 进行反应体系和反应条件优化， 验证方法的特异性， 并通过熔解曲线分析进行虫种鉴别。应用阴性按蚊DNA稀释的间日疟原虫18S rRNA基因重组质粒为模板制作标准曲线， 并分别以质粒DNA和实验室子孢子感染阳性的按蚊DNA为模板分析建立的荧光定量PCR方法的敏感性。在反应体系中加入不同部位、 不同用量按蚊DNA， 以探讨按蚊DNA对检测效果的影响。结果 该方法对按蚊、 人血DNA均无扩增， 对4种疟原虫DNA均有特异性扩增且扩增产物的熔解温度 （Tm） 易于区分， 三日疟原虫、 恶性疟原虫、 卵形疟原虫和间日疟原虫的Tm值分别为71.0、 72.7、 73.9 ℃和75.9 ℃。标准曲线中循环阈值（Ct值） 与模板浓度具有良好的相关性 （相关系数r = -0.99）。最低可以检出含50拷贝的质粒DNA或32倍稀释的子孢子阳性按蚊DNA样本。按蚊DNA对荧光定量PCR反应具有抑制作用。荧光定量PCR的Ct值在实验内和实验间均具有良好的重现性。结论 新建立的SYBR Green I染料荧光定量PCR方法具有较高的敏感性和特异性， 可用于按蚊体内疟原虫子孢子的检测， 并可同时对4种人体疟原虫进行鉴别。

关键词: 荧光定量PCR； 按蚊； 疟原虫子孢子； 虫种鉴别