Abstract：
Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.
摘要：
猪圆环病毒(PCV)已成为一种主要的病原,给全球养猪业造成重大经济损失,PCV2型（PCV2）和3型（PCV3）分布在世界各地。我们设计了针对PCV2Cap和PCV3Rap的特异性引物和探针序列，并在优化引物浓度后开发了多重晶体数字PCR（cdPCR）方法，探针浓度,和退火温度。多重cdPCR检测允许对PCV2和PCV3进行精确和差异检测，检测限为1.39×101和1.27×101拷贝/反应，分别,未观察到与其他猪病毒的交叉反应。测定内和测定间变异系数(CV)小于8.75%,表明良好的重复性和再现性。为了评估该测定法的实用价值，通过cdPCR和定量PCR(qPCR)测试40个组织样品和70个饲料样品的PCV2和PCV3。使用多重cdPCR，PCV2感染率，PCV3感染,合并感染率为28.45%，1.72%，12.93%，分别,并使用多重qPCR，他们是25.00%，0.86%,和4.31%，因此,这种高度特异性和灵敏的多重cdPCR允许精确地同时检测PCV2和PCV3,并且它特别适合于需要用强化处理和复杂基质检测少量输入核酸或样品的应用。
