摘要
目的建立一种简便、快速和高灵敏度的恶性疟原虫的荧光定量环介导等温扩增检测方法。方法针对恶性疟原虫环子孢子蛋白(CSP)种属特异性基因保守区域8个位点设计3对引物,同时使用PCR法扩增恶性疟原虫219bp保守序列,经克隆后转化入工程菌株JM109感受态细胞中并提取重组质粒作为模板。以探索建立恶性疟原虫荧光定量环介导等温扩增检测技术,并对其试验重复性、灵敏度、特异性及适用性予以评估。结果在重组质粒浓度为1.06×104～109拷贝/反应的范围内,标准曲线循环阈值与模板浓度有良好线性关系(相关系数0.9995),CT值变异系数为6.29%,无非特异性扩增,初步临床试验结果表明,试验的灵敏度为90.00%,特异度为93.33%,准确性为91.67%,试验可在30min内完成。结论荧光定量环介导等温扩增技术检测恶性疟原虫简便、快速、敏感,具有广阔的应用前景。
Aim To establish a simple and convenient,quick and high sensitive fluorescent quantitative loopmediated isothermal amplification（FQ-LAMP） for detection of Plasmodium Falciparum. Methods The species conservative regions of P.f CSP gene was selected to design 3 pairs of primers than can recognize 8 distinct sites of the P.f CSP gene,and Plasmodium falclparum＇s recombinant plasmids were constructed as the reaction template,which was used to evaluate the LAMP amplifying efficiency and reliability under different conditions for evaluation the specificity and sensitivity of detection by LAMP. Pneumocystis carinii, Toxoplasma gondii,Plasmodium vivax （P.v）, and healthy human blood DNA were selected as template. The applied value of this method was evaluated by detecting the same batch of 30 artificial infected blood samples, and compared with that of microscopical paralell detection. Results The template concentration was 1.06x104~109 copies per reaction, the standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle （Ct） and template concentration,and the correlation coefficient was 0.9995,and the CV coefficient was 6.29% with no unspecific amplification. Initial clinical trials showed that the sensibility was 90.00% ,specificity was 93.33% and accuracy was 91.67% with a reaction time of within 0.5 hour. Conclusion The method of FQ-LAMP is simple and convenient, high sensitive,which will be a potential method for detecting Plasmodium Falciparum in the public health field.
出处
《中国热带医学》
CAS
2010年第10期1178-1180,共3页
China Tropical Medicine
基金
国家质量监督检验检疫检总局科研专项基金项目(2009IK217)
关键词
环介导等温扩增技术
恶性疟原虫
检测
Plasmodiumfalciparum
Fluorescent quantitative LAMP
Detection
