荧光定量PCR技术评价亚甲蓝光化学法病毒灭活效果的研究

Using Real-Time PCR to Evaluate the Effect of Viral Inactivation by Methylene Blue with Visible Light

本研究探讨利用荧光定量PCR技术评价Sindbis病毒经亚甲蓝光化学处理后灭活效果的可行性。研究采用不同光照强度对Sindbis病毒进行亚甲蓝光化学灭活处理,并用SYBR Green I荧光定量PCR对Sindbis病毒的cDNA进行扩增,同时以细胞病变法做平行对照以测定病毒残余滴度。结果显示在亚甲蓝光化学处理过程中,随着光照强度的增强,病毒残余滴度由6.50 LgTCID50/mL逐渐降低至检测限以下,同时病毒核酸的拷贝数显著下降(P<0.05),并与病毒感染性的降低呈线性相关(R2>0.98)。以上结果表明,亚甲蓝光化学灭活法对Sindbis病毒核酸有破坏作用,病毒核酸损伤程度随光照强度的增强而增加,且与病毒感染性的降低存在相关性,提示荧光定量PCR技术评价亚甲蓝光化学法的病毒灭活效果具有可行性。

To investigate the feasibility of using Real-Time PCR to evaluate the effectiveness of Sindbis virus inactivation by Methylene Blue with visible light. Sindbis virus was treated by Methylene Blue with different intensity of visible light and the transcribed cDNA was quantified by Real-Time PCR. Residual infectivity of treated virus was tested by cell infection method as parallel control at the same time. The residual infectivity of virus decreased from 6.50 lgTCID50/mL to under the limit of detection as light intensity increased. Meanwhile, the quantity of virus cDNA decreased significantly（P（0.05）, which correlated to the decline of virus infectivity（R2）0.98）. Methylene Blue with visible light could cause lesion to nucleic acid of Sindbis virus, the extent of which was light intensity-dependent and correlated to the decrease of virus infectivity. The results demonstrated that Real-Time PCR can be a useful tool for evaluating effect of virus inactivation after Methylene Blue treatment with light.