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Abstract：
Detection of low viral load samples has long been a challenge for African swine fever (ASF) prevention and control. This study aimed to compare the detection efficacy of droplet digital PCR(ddPCR) and quantitative PCR(qPCR) for African swine fever virus (ASFV) at different viral loads, with a focus on assessing the accuracy of ddPCR in detecting low viral load samples. The results revealed that ddPCR had a detection limit of 1.97 (95% CI 1.48 - 4.12) copies/reaction and was 18.99 times more sensitive than qPCR (detection limit: 37.42, 95% CI 29.56 - 69.87 copies/reaction). In the quantification of high, medium, and low viral load samples, ddPCR showed superior stability with lower intra- (2.06% - 7.58%) and inter-assay (3.83% - 7.50%) coefficients of variation than those of qPCR (intra-assay: 8.08%-29.86%; inter-assay: 9.27%-34.58%). Bland-Altman analysis indicated acceptable consistency between ddPCR and qPCR for high and medium viral load samples; however, discrepancies were observed for low viral load samples, where two samples (2/24, 8.33%) exhibited deviations beyond the acceptable range (-46.18 copies/reaction). Moreover, ddPCR demonstrated better performance in detecting ASFV in clinical samples from asymptomatic pigs and environmental samples, with qPCR showing false negative rates of 7.69% (2/26) and 27.27% (12/44), respectively. McNemar analysis revealed significant differences between the two methods (P = 0.000) for samples with a viral load <100 copies/reaction. The results of this study demonstrate that ddPCR has better detection limits and adaptability than qPCR, allowing for a more accurate detection of ASFV in early-stage infections and low-concentration environmental samples. These findings highlight the potential of ddPCR in the prevention and control of ASF.
摘要：
长期以来，低病毒载量样品的检测一直是非洲猪瘟（ASF）预防和控制的挑战。本研究旨在比较液滴数字PCR(ddPCR)和定量PCR(qPCR)对不同病毒载量的非洲猪瘟病毒(ASFV)的检测效果。重点评估ddPCR检测低病毒载量样品的准确性。结果表明，ddPCR的检测限为1.97（95％CI1.48-4.12）拷贝/反应，比qPCR（检测限为37.42，95％CI29.56-69.87拷贝/反应）的灵敏度高18.99倍。在量化的高，中等,和低病毒载量样本，ddPCR显示出较高的稳定性，与qPCR相比，测定内（2.06％-7.58％）和测定间（3.83％-7.50％）变异系数较低（测定内：8.08％-29.86％；测定间：9.27％-34.58％）。Bland-Altman分析表明，对于高和中等病毒载量样品，ddPCR和qPCR之间具有可接受的一致性；然而，在低病毒载量样本中观察到差异，其中两个样品(2/24,8.33%)表现出超出可接受范围的偏差(-46.18拷贝/反应)。此外，ddPCR在无症状猪和环境样本的临床样本中检测ASFV方面表现更好，qPCR的假阴性率为7.69％（2/26）和27.27％（12/44），分别。McNemar分析显示，对于病毒载量<100拷贝/反应的样品，两种方法之间存在显着差异（P=0.000）。结果表明,ddPCR比qPCR具有更好的检测限和适应性,允许在早期感染和低浓度环境样品中更准确地检测ASFV。这些发现强调了ddPCR在预防和控制ASF中的潜力。
