Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China.
College of Food Science and Technology, Hebei Agricultural University, Baoding, China.
Front Cell Infect Microbiol. 2022 Aug 22;12:995705. doi: 10.3389/fcimb.2022.995705. eCollection 2022.
Vascular wilt, caused by and , limits the quality and yield of agricultural crops. Although quantitative real-time PCR (qPCR) has greatly improved the diagnosis of these two pathogens over traditional, time-consuming isolation methods, the relatively poor detection sensitivity and high measurement bias for traceable matrix-rich samples need to be improved. Here, we thus developed a droplet digital PCR (ddPCR) assay for accurate, sensitive detection and quantification of and . We compared the analytical and diagnostic performance in detail of ddPCR and the corresponding qPCR assay against the genomic DNA (gDNA) of the two fungi from cultures and field samples. In our study, the species specificity, quantification linearity, analytical sensitivity, and measurement viability of the two methods were analyzed. The results indicated that ddPCR using field samples enhanced diagnostic sensitivity, decreased quantification bias, and indicated less susceptibility to inhibitors compared with qPCR. Although ddPCR was as sensitive as qPCR when using gDNA from cultures of and , its detection rates using field samples were much higher than those of qPCR, potentially due to the inhibition from residual matrix in the extracts. The results showed that digital PCR is more sensitive and accurate than qPCR for quantifying trace amounts of and and can facilitate management practices to limit or prevent their prevalence.
由 和 引起的维管束萎蔫病限制了农业作物的质量和产量。虽然定量实时 PCR（qPCR）极大地提高了这两种病原体的诊断能力，超越了传统的耗时的分离方法，但对于可追踪的富含基质的样本，其检测灵敏度相对较差且测量偏差较大。因此，我们开发了一种用于准确、灵敏检测和定量 的液滴数字 PCR（ddPCR）分析方法。我们详细比较了 ddPCR 与相应的 qPCR 分析方法针对两种真菌的培养物和田间样本的基因组 DNA（gDNA）的分析和诊断性能。在我们的研究中，分析了两种方法的物种特异性、定量线性、分析灵敏度和测量稳定性。结果表明，与 qPCR 相比，ddPCR 检测田间样本时可提高诊断灵敏度，降低定量偏差，并显示出对抑制剂的敏感性较低。虽然 ddPCR 与 qPCR 一样灵敏，可用于检测培养物中的 和 的 gDNA，但使用田间样本时，其检测率明显高于 qPCR，这可能是由于提取物中残留基质的抑制作用。结果表明，数字 PCR 比 qPCR 更灵敏、更准确地定量痕量的 和 ，并有助于管理实践，以限制或防止它们的流行。
