SYBR GreenⅠ实时荧光定量RT-PCR测定肠道病毒71型(EV71)RNA拷贝数方法的建立

Quantification of EV71 Viral Load Using Real-Time Quantitative RT-PCR with SYBR Green Ⅰ

目的建立SYBR GreenⅠ荧光染料实时定量RT-PCR方法,测定实验动物等来源的EV71病毒RNA。方法运用EV71VP1保守区引物,优化real time RT-PCR条件,运用NASBA方法扩增EV71病毒RNA,计算拷贝数,经10倍系列稀释做出标准曲线,作为EV71病毒RNA定量检测的外标准品。结果应用Qiagen公司QuantiTect SYBR Green RT-PCR Kit,该标准品可精确定量到100copies/μL,PCR扩增效率达到99.5%。结论 SYBRGreenⅠ荧光染料实时定量PCR法测定EV71病毒RNA拷贝数的方法敏感性高、稳定性好,可用于EV71病毒RNA载量的定量测定。

Objective To develop the real-time quantitative RT-PCR technique with SYBR Green Ⅰand to detect the viral load of EV71. Methods The specific primes were used basing on the conserved VP1 and the amplified RNA fragment products by NASBA were accounted into RNA copies as the RNA standards. 10-fold serial dilutions of the RNA standards were quantified using real-time quantitative RT-PCR with SYBR Green Ⅰ and the copy numbers were assessed accordingly. Results From 1 × 108 copies /μL to 1 × 102 copies /μL of 10-fold serial diluted RNA could be quantified with real-time quantitative RT-PCR. The standard curve showed that they had good linear correlation and could be served for the quantification of samples. The amplification efficiency is 99. 5%. Conclusion The RNA standard could be used as an external standard of the real-time quantitative PCR method with SYBR Green Ⅰ. The method has high sensitivity,specificity so that it can be used for quantification of EV71 RNA load in mouse infection models.