鲤疱疹病毒2型微滴式数字PCR检测方法的建立及比较分析

Development and Evaluation of Droplet Digital PCR Assay for the Detection of CyHV-2 and Comparative Analysis

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。

CyHV-2 （Cyprinid herpesvirus 2） is a double-stranded DNA virus first isolated from goldfish, Carassius auratus （L）. It is classified as a member of the genus Cyprinivirus, which includes carp pox （CyHV-1）, koi herpesvirus （CyHV-3） and anguillid herpesvirus-1 （AngHV-1）. It has an icosahedral shape with an average diameter of 100–110 nm. CyHV-2 can cause approximately 50% to 100% mortalities in cultured goldfish when water temperature is between 15℃ and 25℃. Therefore, rapid and precise detection of CyHV-2 is needed to prevent and control disease outbreak. In this study we established a droplet digital PCR （ddPCR） method used for accurate quantification of CyHV-2 DNA. The ddPCR method was compared with the quantitative real-time PCR （qPCR） in the aspect of the sensitivity, reproducibility, specificity and practical application. In terms of detecting CyHV-2 DNA, the sensitivity of the ddPCR assays was 20-fold lower than qPCR. The correlation coefficient （R2） obtained from linear regression analysis showed a good linearity of amplification for both ddPCR （R2=0.994） and qPCR （R2=0.994） assays. A positive correlation （R2=0.989） was observed between ddPCR and qPCR assays. To determine the same dilution series of CyHV-2 DNA, the expected numbers of DNA copies calculated by qPCR was 10-fold higher than the number of copies determined by ddPCR. CyHV-2 DNA reproducibility determined by ddPCR was found to be significantly more stable than by qPCR. The ddPCR assay had no cross-reaction with other similar fish herpesviruses, including CyHV-3, CCV （Channel catfish virus）、STIV （Soft-shelled turtle iridovirus） and EHNV （Epizootic hematopoietic necrosis virus）. By contrast, the specificity of ddPCR was consistent with qPCR. Therefore, the ddPCR method was proven to be more precise than qPCR. This new absolute quantitation tool will be useful to standardize quantitative detection of CyHV-2 DNA.