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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 PCR (dPCR) is a new method for absolute quantification of nucleic acids. However, the absolute quantification method of influenza A virus (FluA) based on dPCR has not been established systematically. In this study, we optimized the annealing temperature of microdroplet dPCR firstly, and determined the best annealing temperature of dPCR reaction was 64.4 ℃. Using FluA nucleic acid standard, the detection range of FluA for microdroplet dPCR was 37.7 ~ 8.22 ”104 copies / # L, the detection limit was 3.77 copies / reaction.The correlation coefficient between the detection results of microdroplet dPCR and the standard copy number is R2 = 0.9988, which indicates that the test results of the method are highly credible Degree.Under the established microdroplet dPCR method, we can quantify the copy number of FluA in clinical samples.Therefore, FluA absolute quantification method based on microdroplet dPCR was established in this study, The absolute quantification of influenza viral load provides a technique for the determination of viral load in clinical studies.