一种检测慢病毒滴度的实时荧光定量PCR方法  

A Real-Time Fluorescence Quantitative PCR Method for Detecting Lentivirus Titer

作　　者：张飞飞 孙文 耿琦 丁怡彤 ZHANG Fei-Fei;SUN Wen;GENG Qi;DING Yi-Tong(College of Anesthesiology,Xuzhou Medical University,Jiangsu Province Key Laboratory of Anesthesiology,Xuzhou 221004,China)

机构地区：[1]徐州医科大学麻醉学院江苏省麻醉学重点实验室

出　　处：《生物技术通讯》2019年第4期523-527,588,共6页Letters in Biotechnology

基　　金：江苏省高等学校自然科学研究(17KJD180006);徐州医科大学校级课题(2017KJ08)

摘　　要：目的:建立一种有效、灵敏、准确的慢病毒滴度的分子检测方法。方法:分别构建慢病毒调控元件WPRE和单拷贝基因白蛋白(Alb)基因的重组质粒,紫外吸收法测量后经数学换算得到相应的拷贝数,以梯度稀释质粒为模板,利用基于SYBR Green的荧光定量PCR制作标准曲线,最后将待测样品的Ct值代入标准曲线,根据相应公式计算慢病毒滴度。结果:WPRE元件和Alb基因质粒的标准曲线回归方程分别为y=-4.255x+46.047、y=-2.8735x+35.831,相关系数R2均大于0.99,扩增效率E均大于95%,且熔解曲线波峰单一。计算获得不同稀释比例待测病毒的滴度为(4.3±0.9)×10^6 TU/mL。结论:基于SYBR Green的实时荧光定量PCR方法操作简便,能够准确测定慢病毒滴度。Objective:To establish an effective,sensitive and accurate molecular detection method for lentivirus titer.Methods:Recombinant plasmids of lentivirus regulatory element WPRE and single copy gene albumin were constructed respectively.After UV absorption measurement,the corresponding copy number was obtained through mathematical conversion.Standard curves were prepared by quantitative fluorescence PCR based on SYBR Green using gradient dilution plasmid as template.Finally,the Ct value of the samples was substituted into the standard curve,and the lentivirus titer was calculated according to the corresponding formula.Results:The standard curve regression equation of WPRE element and Alb gene was y=-4.255x+46.047 and y=-2.8735x+35.831,respectively,R2>0.99,amplification efficiency>95%and the wave peak of melting curve was single.The titer of the virus with different dilution ratios was calculated to be(4.3±0.9)×10^6 TU/mL.Conclusion:The real-time fluorescence quantitative PCR method based on SYBR Green is simple and can accurately measure the lentivirus titer.

关 键 词：慢病毒滴度 实时荧光定量PCR WPRE 白蛋白基因 

分 类 号：Q78[生物学—分子生物学]