猪圆环病毒3型微滴数字PCR定量检测方法的建立

Development of Droplet Digital PCR for Quantitative Detection of Porcine Circovirus Type 3

摘要:

猪圆环病毒3型是一种新型猪圆环病毒，可引起猪感染相关疾病。本实验根据猪圆环病毒3型的OPF2基因序列，设计引物探针，建立了可对其准确定量检测的微滴数字PCR(droplet digital PCR，ddPCR)方法。通过对ddPCR反应体系中的引物探针浓度和退火温度进行优化，得到ddPCR反应的最佳引物探针浓度比为800 nM: 800 nM: 400 nM；最佳退火温度为60 ℃。该ddPCR方法的标准曲线相关系数(R2)为0.999，呈良好的线性关系；灵敏度高，可达4.4 copies/μL；特异性良好，与常见猪病原无交叉反应。通过对临床样品的检测结果证明，本研究建立的猪圆环病毒3型ddPCR检测方法比实时荧光PCR检测方法的灵敏度高1个数量级，检测结果与荧光PCR定性结果一致，并且可对可疑样本进行定量分析。结果表明：新建立的ddPCR方法灵敏度高、特异性强，可用于猪圆环病毒3型的定量检测。

Abstract:

Porcine circovirus type 3 is a novel porcine circovirus that can cause diseases associated with porcine infection. In this experiment, primer probes were designed based on the OPF2 gene sequence of porcine circovirus type 3, and a droplet digital PCR (ddPCR) method for accurate and quantitative detection was established. After optimization of the primer probe concentration and annealing temperature for the ddPCR reaction system, the optimal primer probe concentration ratio was 800 nM: 800 nM: 400 nM; and the optimal annealing temperature was 60 ℃. The correlation coefficient R2 of the ddPCR standard curve was 0.999, showing a good linear relationship. The sensitivity was high with the limit of detection being 4.4 copies/μL and good specificity (no cross-reaction with the common pig pathogens ). The detection results of the clinical samples revealed that the ddPCR method established in this study was more sensitive than the real-time fluorescent PCR method by one order of magnitude, and could generate results consistent with those obtained by the fluorescent PCR and analyze quantitatively suspicious samples. The results showed that the newly established ddPCR method in this study had high sensitivity and specificity, thus could be used for quantitative detection of porcine circovirus type 3.