基于微滴式数字PCR技术的甲型流感病毒绝对定量方法的建立及应用

Droplet Digital Polymerase Chain Reaction Method for Absolute Quantification of Influenza A Viruses

数字PCR(Digital PCR,dPCR)是核酸绝对定量的新方法,然而,基于dPCR的甲型流感病毒(Influenza A virus,FluA)绝对定量方法还未系统建立。本研究首先对微滴式dPCR的退火温度进行了梯度优化,确定了dPCR反应的最佳退火温度为64.4℃;利用FluA核酸标准品,确定了微滴式dPCR对FluA的检测范围为37.7~8.22"104拷贝/#L,检测的检出限为3.77拷贝/反应。微滴式dPCR的检测结果与标准品拷贝数的相关系数为R2=0.9988,提示该方法检测结果具有较高的可信度。用建立好的微滴式dPCR方法可对待测临床样本中的FluA进行了拷贝数定量。因此本研究建立了基于微滴式dPCR的FluA绝对定量方法,可有效地对临床样本中甲型流感病毒载量进行绝对定量,为临床研究中病毒载量的测定提供了一种技术。

Digital polymerase chain reaction （dPCR） is a new method for absolute quantification. However, an absolute quantification method for influenza A viruses based on droplet digital polymerase chain reaction （dPCR） has not been established. In this study, we found that： （i） the annealing temperature of dPCR was optimized at 64.4℃; （ii） the detection range of dPCR was 37.7-8.22× 10^4 copies/μl for influenza A viruses; （iii） the limit of detection of dPCR was 3. 77 copies/reaction. The liner correlation coefficient （R2） was found to be 0. 9988, suggesting the high reliability of our dPCR method. The dPCR method could be used to detect influenza A viruses clinically. In summary, we developed a dPCR method for absolute quantification of influenza A viruses. This method could be used effectively to quantify influenza A viruses in clinical samples. Therefore, our method could be a new tool for the absolute quantification of viral load.