摘要
目的建立对4种感染人的疟原虫虫属及定量的环介导等温扩增法(loop-mediated isothermal amplification,LAMP)基因检测方法。方法根据恶性疟原虫、三日疟原虫、卵形疟原虫、间日疟原虫的18SrRNA基因共有保守序列及LAMP技术原理,设计一套含有6条疟原虫虫属特异性引物的LAMP反应体系,产物经SYBR GreenⅠ进行显色反应及电泳分析,观察其特征条带的情况,利用LAMP技术检测上述4种疟原虫DNA及其它12种相关寄生虫DNA评价反应特异性,利用PCR技术为参照对LAMP技术检测疟原虫虫属试验的敏感性,利用疟原虫重组质粒探索建立疟原虫LAMP定量的可能性。结果 4种疟原虫LAMP产物经显色后呈绿色,经电泳后呈特征性梯状条带,阴性对照及其它相关寄生虫DNA无明显扩增,重组质粒浓度在1.42×104～1.42×109拷贝/μl范围内时,反应循环阈值与模板浓度有良好的线性关系(r=0.9995)。与PCR方法相比较,建立的疟原虫虫属LAMP检测方法的敏感性是其10倍,检出限达到1.42×101拷贝/μl。结论建立的检测疟原虫属种、数量的LAMP方法具有较高的特异性和敏感性,适合疟疾防治检测的需要。
Objective To develop a quantitative detection method using loop-mediated isothermal amplification （LAMP） to detect Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. Methods Based on the conserved nucleotide of the four species and the mechanism LAMP, a LAMP reaction system containing six primers was designed for genus detection and quantitation. LAMP products were stained by SYBR Green and analyzed by electrophoresis. The specificity of LAMP was evaluated by detection of Plasmodium spp. and twelve species of related parasites. The sensitivity of LAMP was evaluated in comparison with the PCR sensitivity, and the feasibility of quantitative LAMP was evaluated by detection of recombinant plasmids. Results After staining, LAMP products with Plasmodium DNA turned green while the color of the negative control and other parasites remained unchanged. Electrophoresis showed that LAMP products had characteristic ladders, which was not present in the negative control and other parasites. At a template concentration of 1.42×10^4-1.42×10^9 copies per microlitre, the standard curve established by recombinant plasmids showed a fine linear relationship between threshold cycle （Ct） and template concentration, with a correlation coefficient of 0.9995. The LAMP assay was found to have a detectability of 1.42×10^1 copies/μ1, a 10-fold increase in sensitivity compared with traditional PCR. Conclusion The detection system had high specificity and sensitivity, which could be used for the prevention and control of malaria.
出处
《中国媒介生物学及控制杂志》
CAS
CSCD
2011年第4期355-358,共4页
Chinese Journal of Vector Biology and Control
基金
国家质检总局科研专项基金(2009IK217)~~
