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本研究建立了定量检测鲤疱疹病毒2型(Cyprinid herpesvirus 2,CyHV-2)的微滴式数字PCR(Droplet digital PCR,ddPCR)检测方法,并与实时荧光定量PCR(Quantitative real-time PCR,qPCR)检测方法的灵敏性、重复性、特异性和临床样品检测做了比较分析。结果表明,与qPCR相比,ddPCR具有相同的特异性,其灵敏性比qPCR低20倍。在定量CyHV-2 DNA时,ddPCR(R~2=0.994)和qPCR(R~2=0.994)均表现出良好的线性关系,且2种检测方法间的定量值呈正相关(R~2=0.989)。在定量检测相同稀释度的CyHV-2 DNA时,qPCR的定量值始终比ddPCR高10倍。ddPCR的组内和组间重复变异系数(CV)分别为0.59%–11.26%和6.55%–23.21%,而qPCR为16.57%–27.56%和22.31%–56.73%,说明ddPCR具有更好的稳定性。在临床样品定量检测时,ddPCR的检出率稍高于qPCR。本研究建立的ddPCR能够准确定量检测CyHV-2,将为CyHV-2相关研究提供有益参考。
In this study, we established a droplet digital PCR (ddPCR) method for the quantitative detection of Cyprinid herpesvirus 2 (CyHV-2) and compared it with real-time quantitative PCR (qPCR The sensitivity, repeatability, specificity and detection of clinical samples were comparatively analyzed. The results show that ddPCR has the same specificity as qPCR with a sensitivity 20-fold lower than qPCR. There was a good linear relationship between ddPCR (R ~ 2 = 0.994) and qPCR (R ~ 2 = 0.994) in quantifying CyHV-2 DNA, and the correlation between the two detection methods was positive (R ~ 2 = 0.989 ). The quantitative value of qPCR was always 10 times higher than that of ddPCR when quantitatively detecting CyHV-2 DNA of the same dilution. The intra-and inter-group repetitive coefficient of variation (CV) of ddPCR were 0.59% -11.26% and 6.55% -23.21%, while qPCR was 16.57% -27.56% and 22.31% -56.73%, respectively, indicating that ddPCR has better stability . The detection rate of ddPCR was slightly higher than that of qPCR when the clinical samples were quantitatively detected. The ddPCR established in this study can accurately and quantitatively detect CyHV-2, which will provide a useful reference for CyHV-2 related research.