猴痘病毒微液滴数字PCR检测方法的建立

Establishment of droplet digital PCR detection method for mpox virus

目的建立一种用于猴痘病毒(mpox virus)核酸检测的微液滴数字PCR(droplet digital PCR,ddPCR)检测方法。方法设计靶向猴痘病毒F3L基因的特异性引物和探针,建立猴痘病毒核酸的ddPCR检测方法,对ddPCR的最适反应条件进行优化,并对ddPCR的特异性、灵敏度以及重复性进行检测。进一步对人脓拭子临床样本进行检测,对ddPCR方法检测临床样本的能力进行评价。结果对ddPCR反应温度和引物探针浓度进行优化,建立猴痘病毒ddPCR检测方法,该方法最低检测限可达2拷贝/μl,与其他8种病毒无交叉反应。对10份人皮肤脓液拭子临床样本中的猴痘病毒进行检测,ddPCR方法检测到5份阳性样本,实时荧光定量PCR(real time fluorescence quantitative PCR,qPCR)方法检测到4份阳性样本。结论建立的猴痘病毒的ddPCR检测方法快速、特异、灵敏度高,用于临床样本中猴痘病毒检测。

Objective To establish a droplet digital polymerase chain reaction(ddPCR)detection method for monkeypox(mpox)virus.Methods Specific primers and probe targeting the F3L gene of mpox virus were used to develop a ddPCR detection method for the mpox virus′s nucleic acid.The ddPCR reaction conditions were optimized,and the specificity,sensitivity,and repeatability of ddPCR were assessed.Additional examination of clinical samples obtained from human pus swabs was performed and the ddPCR method′s ability to detect clinical samples was evaluated.Results The result demonstrated that the mpox virus ddPCR detection method was established and the ddPCR reaction temperature and primer probe concentration has been optimized.The method had a minimal detection limit of 2 copies/μl and did not exhibit cross reaction with the remaining 8 viruses.Ten clinical samples of human skin pus swabs were tested for the mpox virus;five of the samples were tested positive by ddPCR,and four of the samples were tested positive by real time fluorescence quantitative PCR(qPCR).Conclusions The established ddPCR for mpox virus is a fast,specific,and highly sensitive detection method,and can be used for the detection of mpox virus in clinical samples.