登革1型病毒荧光定量PCR快速检测

Development of real-time PCR for detection of dengue virus type 1

目的建立登革1型病毒的快速特异的荧光定量PCR检测方法。方法设计登革1型病毒特异的引物和TaqMan探针,制作标准曲线,并检测其特异性、重复性、再现性和敏感性,采用荧光定量PCR和常规PCR对临床标本进行检测比较。结果成功构建了标准曲线,回归方程为Y=-3.410logX+45.10,相关系数R2=0.999,扩增效率Eff=96.5%,灵敏度可达103 copies/mL,此荧光定量PCR方法对登革1型病毒检测高度特异,与其他3型登革病毒和乙型脑炎病毒之间并无交叉反应;采用登革病毒种特异引物探针和本研究建立的登革1型病毒型特异引物探针对166份cDNA样本进行荧光定量PCR检测,均发现126份为阳性,阳性率均为75.9%,Ct值为20.84～36.36,浓度范围为1～1.3×104copy/μL;常规PCR方法检测到其中36份为阳性,阳性率为21.7%。结论荧光定量PCR方法能够快速灵敏地检测登革1型病毒,并能实现对病毒滴度的绝对定量。

Objective To develop a real-time PCR method for the detection of dengue viruses. Methods Multiple alignments of dengue virus sequence were designed and used to develop a system of real-time PCR to detect dengue virus type 1 strain. The system was validated for specificity, sensitivity, repeatability, and reproducibility and then applied to a series of human samples. Results The specificity of the system was determined by experimentall tests on different flavivir- uses. There were no cross reactions with Japanese encephalitis virus（JEV） and other serotypes of dengue virus. The system allowed the detection of less than one infectious particle and was able to detect dengue virus type 1 in human samples where infectious virus cannot be isolated. Conclusion The system developed is valuable for rapid detection of dengue virus type 1 and epidemiological studies.