检测基孔肯雅病毒SYBR Green I real-time PCR方法的建立及应用

Establishment of a SYBR Green I real-time PCR technique and its use to detect Chikungunya virus

目的建立一种基孔肯雅病毒的快速诊断方法。方法采用基孔肯雅病毒E1基因的重组质粒作为阳性标准品。优化反应条件,建立针对基孔肯雅病毒的SYBR Green I real-time PCR检测方法,并对其特异性、灵敏性和重复性进行评价。结果建立的SYBR Green I real-time PCR检测方法的Ct值与阳性标准品在6.94×10~0~6.94×10~8 copies/μl范围内呈良好的线性关系,相关性为0.997,斜率为-3.571;其检测下限为0.694 copies/μl,且对ZIKV、DENV和JEV等均无特异性扩增;所有稀释倍数的标准品在84.0℃出现特异性熔解峰;组内和组间变异系数均小于2%。采用该方法对93份蚊虫样品进行检测,基孔肯雅病毒核酸阳性率为2.15%,普通PCR核酸阳性率为1.07%。结论成功建立了针对基孔肯雅病毒E1基因的SYBR Green I real-time PCR检测方法。该方法灵敏、特异,可用于基孔肯雅病毒感染的快速诊断。

Objective To establish a method for rapid diagnosis of the Chikungunya virus.Methods A recombinant plasmid of the Chikungunya virus E1 gene was used as the positive standard.SYBR Green I real-time PCR was optimized to specifically detect Chikungunya virus,and its specificity,sensitivity,and reproducibility were tested.Results Results indicated a good linear relationship between the Ct values of the devised technique and those of the positive standard in the range from 6.94×10~8 to 6.94×10~0 copies/μl;the correlation was 0.997 with a slope of-3.571.The detection limit was 0.694 copies/μl,and the devised technique did not amplify ZIKV,DENV,or JEV.Standard templates in various dilutions all displayed specific melting peaks at 84.0 C.The devised technique detected Chikungunya virus nucleic acids in mosquito samples at a rate of 2.15%while normal PCR did so at a rate of 1.07%.Conclusion A technique for SYBR Green I real-time PCR to detect the E1 gene of the Chikungunya virus was successfully established.This technique is sensitive,specific,and can be used for rapid diagnosis of a Chikungunya virus infection.