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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。
Objective To establish a fluorescence quantitative PCR method for the quantitative detection and species identification of Plasmodium sporozoites in Anopheles. Methods A pair of primers specific for the 18S rRNA genes of four human malaria parasite strains was used to optimize the reaction system and reaction conditions by using a mixture of Plasmodium falciparum 18S rRNA gene recombinant DNA and Anopheles sinensis DNA as a template to verify the specificity of the method Sex, and by melting curve analysis of species identification. The standard curve of the 18S rRNA gene recombinant plasmid of Plasmodium vivax was used as the template to establish the standard curve and the sensitivities of the established real-time quantitative PCR method were analyzed respectively using plasmid DNA and laboratory positive sporozoite DNA as template . In the reaction system by adding different parts, different doses of Anopheles DNA, to explore Anopheles DNA on the detection effect. Results The method did not amplify the DNA of Anopheles sinensis and human blood, and amplified the DNA of all the four Plasmodium species. The melting temperature (Tm) of the amplified product was easy to distinguish. Plasmodium malariae, Plasmodium falciparum, The Tm values of P. falciparum and P. vivax were 71.0, 72.7, 73.9 and 75.9 ° C, respectively. The standard curve of the cycle threshold (Ct value) and the template concentration has a good correlation (correlation coefficient r = -0.99). A minimum of 50 copies of plasmid DNA or a 32-fold diluted sporozoite positive Anopheles DNA sample can be detected. Anopheles DNA can inhibit the fluorescence quantitative PCR reaction. Fluorescent quantitative PCR Ct values in the experimental and experimental have good reproducibility. Conclusion The newly established SYBR Green I dye fluorescence quantitative PCR method has high sensitivity and specificity. It can be used for the detection of Plasmodium sporozoites in Anopheles mosquitoes and can identify four kinds of human Plasmodium.